Immunological methods of treating cancer

ABSTRACT

A drug, affecting the hyperactivated immunologic effector cells, comprising (I) a Ca-antagonist and (II) an agent, reducing the intracellular cAMP/cGMP-ratio, is being described. In addition, a drug, affecting the hyperactivated immunologic effector cells, consisting of (I) an agent, eliminating the hyperactivated effector cells and (II) alloreactive cells with predetermined cell death, is being described.

BACKGROUND

[0001] The invention deals with an agent, able to affect thehyperactivated immununological effector cells and with its use.

[0002] Different situations and disorders of humans are associated witha hyperactivated state of the immunological-effector cells, caused e.g.by cytokines; such effector cells lose their ability to respond to newspecific signals. The immune system is impaired or even switched off insuch situations. This occurs e.g. following a persistent stimulationduring a prolonged infection or in situations of cell hyperactivationdue to an excessive release of endogenous cytokines. The term“immunological effector cells” comprises e.g. T cells,macrophages/monocytes, NK cells and other immunological cells.

[0003] A variety of immunological processes include on the cellularlevel the cyclic adenosine phosphate (cAMP) which is produced by theenzyme adenylate cyclase (AC) from adenosine triphosphate (ATP). ThecAMP plays as “second messenger” a central role in the hormonalregulation as well as in the metabolism (through activation of proteinkineses, e.g. protein kinase A (PKA). The PKA phosphorylates proteinswhich in turn depress the immune response. In this way, thehyperactivation of effector cells results in the down regulation of theimmune function.

[0004] This reaction cascade is regulated by the production of cyclicguanosine monophosphate (cGMP) which antagonizes the cAMP. This reactioncascade is also influenced by the group of G-protein coupled receptors,comprising receptors such as adrenergic, muscarinic, histamine,serotonin and adenosine receptors. The G-proteins (guaninenucleotide-binding proteins) are able to stimulate (Gs) or to inhibit(Gi) the production of second messengers. By affecting either the Gs- orthe Gi-receptors, the stimulation of AC and herewith the cAMP-productioncan be regulated. Situations with a disturbed equilibrium are e.g.cancer, viral diseases and autoimmune disorders, as well as the inducingand disease-maintaining component of the atherosclerosis.

SUMMARY

[0005] The objective of the invention was to provide an agent, able tofight with such disorders and to restore the susceptibility of thehyperactivation-depressed immune system for signals and a normal immuneresponse. This objective can be achieved by an agent, capable to affectthe hyperactivated immunological effector cells which comprises (I) aCa-antagonist, and (II) an agent, able to decrease the intracellularcAMP/cGMP-ratio. Surprisingly, it could be found out that such acombination reduced or prevented the hyperactivation of effector cells.In this way, these cells re-acquire their susceptibility for specificsignals and show a normal immune reaction. It could be shown thathyperactivated cells contain an excess of calcium ions and arecharacterized by an increased cAMP/cGMP-ratio.

[0006] According to the invention, the principle of the agent is theprevention of Ca²⁺-influx and reduction of cAMP/cGMP-ratio in immuneeffector cells. This can be achieved by the combination of component Iand II. Surprisingly, in this way, diseases as different as cancer,autoimmune disorders, arteriosclerosis which seems to need an autoimmunepromotor for its provocation, further bacterial, viral, including retroviral infections, as well as some “modern” diseases, based onimmunological derailment or deviations, e.g. the CFS (chronic fatiguesyndrome) can be treated. All these situations and disorders appear tocontain—in the immunological sense—common disregulation elements. Thesecommon elements are e.g. a persistent or excessive activation of certainleukocyte-subpopulations, mostly macrophages and helper T cells, as wellas suppressor cells. During a simultaneous hyperactivation ofmacrophages and helper T cells, as observed in HIV-patients, a mutualstimulation of both interdependent leukocyte-subpopulations can occur.

[0007] A further common element is the deblockade of blastogenicallypretransformed T4 and plasma (B) cells as consequence of preceding,persistent latent or manifest immunosuppression; it is based on a“critical drop” of autoantigen or pathogen-specific surveyor cells orsuppressor T cells. From the molecular-biologic point of view, thepersistence of an increased intracellular Ca_(i) ²⁺-level, leading to animmunosuppressive counter regulation and to an impaired sensitivity tonew antigenic signals, is a common element of a variety of disordersmentioned above. This results not only in the impairment of the existentset of immunocompetent cells but, in addition, in a disturbedrecruitment of new, intact immunocytes.

[0008] The combination of components I and II, according to theinvention, deblocks the misprogrammed immunocytes or effector cells,primarily those in the hyperactivated state, and has therefore impact onthe “wound healing” or “tissue repair”—function of misprogrammedmacrophages and cytokine-hyper secreting helper T cells. Thishyperactivated or persistently activated state of effector cells can beprevented through component I by breaking the Ca²⁺-rigidity, i.e. bybreaking the Ca_(i) ²⁺-overload of effector cells; on the other hand,the Ca_(i) ²⁺-level is regulated by the intracellular pHi. An additionalmechanism is the control of the electrolyte transport through the cellmembrane, i.e. the regulation of the Ca²⁺-, Na⁺- and K⁺-channels and ofthe Na⁺/K⁺-, Na⁺/H⁺-, K⁺/H⁺-, HCO₃-/CI- and Ca²⁺/Na⁺-antiport, symportand different ATPases, respectively. According to the invention, thecombination was able to reduce the primary tumor and metastases incancer patients by {fraction (1/3)} to ⅔ within few weeks, without asimultaneous radio- or chemotherapy.

[0009] The CFS-patients also showed surprising therapeutic results. Herepresented examples of component I and II can be combined as well. Theireffect in patients with neoplastic and autoimmune diseases,atherosclerosis, amyloidosis, Alzheimer disease and CFS can further bepotentiated by combining them with known immunostimulators and BRMs. Inaddition, these combinations can be used therapeutically in bacterialand viral infections. Herewith, the initial derailment, i.e. thehyperactivation of leukocyte-subpopulations can be reversed. Therestoration of the original immune state then occurs spontaneously. Thetherapeutic protocol can consist of 2 or 3 steps. So, in the first phasea deblockade of immunocompetent, misprogrammed effector cells, primarilymacrophages and T cells, can occur, and in the second phase, acontrolled stimulation of immunocytes can follow.

[0010] In a further modification, a “freezing-up” of the controlledstimulated cell state, i.e. a prolongation of the second phase (phase 3)is foreseen. In the first phase, the drug combination, according to theinvention, is used. In the second phase, well known immunostimulatorsand in the third phase, the same combination as in the first phase, butat reduced concentration, e.g. 20 to 50% of the concentration, used inthe first phase, are foreseen.

DETAILED

[0011] Details about the deblocking of immunocompetent cells in hypoxicand/or acidified milieu-of chronically inflammated tissues, includingnecrotic tumor tissue are described.

[0012] Hyper- or persistently activated macrophages consume up to 10-20times more oxygen than resting macrophages; in the presence ofgamma-interferon, this O₂-consumption increases additionally.

[0013] The phagocytosis of opsonized particles (bacteria, latex etc.) aswell as of immune complexes via the Fc-gamma-receptor switches on theEmbden-Mayerhof-glycolysis pathway. Herewith, the generation of themicrobicidal oxygen radicals (ROI) from molecular oxygen is stimulated.In addition, the synthesis of leukotriens C, D and E (via reducedglutathione) is impaired due to a direct electron and proton transfer onO₂-radicals (mediated by cytochrome P450). Leukotrien B which issynthesized instead of the leukotriens C, D and E stimulates directlythe cytochrome P450-mediated ROI-generation.

[0014] The glycolysis leads via the excessive production and secretionof lactic acid to a strong acidification of the macrophage micro milieu.The pH-drop results in the suppression of immunocyte function. Theimpaired synthesis of Leukotrien C, D and E, additionally affected byROI, results in an inhibited glycosylation and secretion of cytokinesand immunoglobulins.

[0015] The hypoxia inflammation tissue, e.g. necrotic tumor tissue,caused by the impaired angiogenesis and hyperactivated macrophages leadsto pH-drop which results in cell depression (below pH 6,8) and celldeath at a continued pH-drop.

[0016] Both, the apoptotic and the autolytic cell death appear to arisefrom the drop of intracellular pH which is associated with theactivation of lysosomal enzymes. A defect in the H⁺-pump which maintainsa lysosomal pH of 4.5-5.0 precedes the cell death.

[0017] As known from the myocard cells, the cell death is accelerated inthe presence of oxygen as terminal electron acceptor if the cell isactivated and herewith the intracellular Cai-level is increased.Therefore, under hypoxic or ischemic conditions, the glucose catabolismvia the TCC/citrate cycle should be inhibited by special drugs.Herewith, the overproduction of NADH₂ (and NADPH₂) which leads to anaccelerated glycolytic lactate synthesis and to pH drop, can beprevented. For this reason, BRMs have to be combined with deblockingsubstances, or the deblocking of immunocompetent cells has to precedeother (immuno)therapeutic steps. The component II comprises an agentwhich is able to affect the cAMP/cGMP-ratio. One variant proposes theincrease of intracellular cGMP-level.

[0018] One of the main reasons for the blocked function ofimmunocompetent cells (mononuclear and polymorphonuclear phagocytes, NKcells, K cells, T cells) in situ/in vitro e.g. in tumor lesions or inchronic inflammation tissues is the low cGMP-level or the increasedcAMP-level in the cytosol of immunocompetent cells.

[0019] The increased cAMP-level arises primarily from the activation ofadenylate cyclase by hyperproduced prostaglandins (PGE2/El); theseprostaglandins are secreted by hyperactivated macrophages, fibroblastsand synovial cells. They activate, together with catecholarnines, theenzyme adenylate cyclase which results in cAMP-increase in cytosol.

[0020] The intracellular cAMP-rise inhibits different cell functions ofvarious immunocyte subclasses. Therefore the induction of guanylatecyclase and herewith the cGMP-rise in immunocompetent cells isrecommended according the invention. In this way, the blocked(preinactivated) effector cells switch their function from thesuppressor to the effector.

[0021] According to the invention, the following agents can be usedtherapeutically (a) as single agents, or (b) combined with each other,or (c) combined as single agents or as mutual combinations (see (b))with different BRMs:

[0022] (1) alkalizing substances, such as alkali-(bi)carbonate andalkali-salts of metabolizable organic acids (e.g. Nalactate,Na-gluconate, Na-, K-citrate)

[0023] (2) reversible competitive inhibitors of citrate oxidation inKrebs/citrate cycle, i.e. inhibitors of tricarboxylic acids in citratecyclus (e.g. tricarballylate, methyl/ethyl-succinate, malonate etc.).The working mechanism is the prevention of cell (e.g. macrophage)hyperactivation in situations, associated with O₂-deficit.

[0024] (3) β-blockers in general. The working mechanism is theinhibition of intracellular cAMP-rise and of A-kinase (PKA) -activity.

[0025] (4) Ca-antagonists in general. The working mechanism is theinhibition of Ca²+-influx into the hyperactivated (hypoxic) cell whichprevents the Ca_(i)-stimulated oxidation in Krebs cycle duringO₂-deficit and herewith the cell death. In the case of hyperactivatedmacrophages, the ROI generation can be inhibited in this way. These ROIsblock in turn the glycosylation and secretion of cytokines.

[0026] (5) Ca-agonists in general. The working mechanism is like in thecase of β-blocker (point (3)), the Ca_(i)-increase. The following2-step-protocol is recommended: first, the activity of the“misprogrammed”, i.e. hyperactivated macrophages has to be blocked byCa-antagonists (point (4)); the 2^(nd) step is the activation ofimmunocompetent cells by Ca-agonists and/or β-blockers.

[0027] (6) Substances, preventing phi-drop by penetrating into the celland binding the excessive H⁺-ions. This class of substances comprisesall representatives of alkaline compounds, e.g. derivatives of TRIZMA,HEPES, mono-, di- and triethanolamines, as well as chloroquine and otherantimalaria drugs, further monensin and compounds which are able torelease NH₃ intracellularly, such as glutamine and asparagine. FurtherpH_(i)-increasing substances are LI-, Cs- and Rb-salts of carbonic acidand organic acids. The weak bases have to be used as free bases, as(bi)carbonate or as salts of metabolizable organic acids, but not aschloride, sulfate, nitrate etc. These substances can be combined withalkali-salts of metabolizable organic acids (see point (1)) and/or withinhibitors of TCC/citrate cycle.

[0028] (7) Inhibitors of LDH (lactate dehydrogenase) and XOD (xanthinoxydase). The working mechanism is the inhibition of lactate productionwhich prevents the excessive acidification of cytosol and extracellularmilieu. These LDH- and XOD-inhibitors can be combined with inhibitors ofTCC/Krebs-cycle.

[0029] (8) Substances, able to correct the intracellularredox-potential, e.g. fumarate/maleinate, vitamin .C, vitamin A, vitaminE, alkali (K)-ferrocyanide, Se-compounds (e.g. Na-selenite),Na-/K-thiosulfate, alkali-sulfate and compounds carrying reduced formsof mercaptylt/thionyl (-SH)-groups, e.g. glutathione, penicillamine,thiola(thiopronine), cysteine, methionine etc.

[0030] (9) Substances, correcting the intracellular NAD(P)⁺/NAD(P)H₂- orthe GSSG/2GSH-ratio, e.g. N-acetyl cysteine.

[0031] (10) Substances, replacing the terminal electron-acceptor(molecular O₂) of the oxidative phosphorylation, e.g. ascorbate,dehydroascorbate, insaturated fatty acids.

[0032] (11) ROI-scavengers and/or antioxidants, e.g. (a) phenols such astocopherols, flavonoids, phenolic acid plus esters, benzodioxols,lignanes (NDGA), BHT, BHA, THBP (b) amines such astetramethyl-p-phenylenediamine (c) heterocyclic compounds such asethoxyquinine, barbiturates, carbazols, phenothiazines, levamisole,nafazatron, naloxone and tinoridine (d) different compounds such asvitamin C, glutathione (GSH), β-carotine and vitamine A-derivatives.

[0033] (12) Cl⁻-channel blockers

[0034] (13) Cyclooxygenase inhibitors/NSAIDs

[0035] (14) H₂-specific antihistaminics (antagonists of the H₂-histaminereceptor), e.g. cimetidine

[0036] (15) inhibitors of the cAMP- and cGMP-phosphodiesterase(methylxanthines; e.g. theophylline or theobromine

[0037] (16) Since the immune complexes (IC) suppress—via specialreceptors, such as the Fc(gamma)-R and/or CR1, CR3, CRq and otherreceptors—both the macrophages/monocytes and NK cells and stimulatesuppressor T cells (T_(G)/Ts), the IC interaction with immunocytereceptors has to be blocked, according to the invention, (a) by theFc(gamma)-subunit of Ig (IgG), (b) by the biotechnologically modifiedcomplement subunits (replacement of key amino acids), e.g. c3b, c3bi orclq and/or (c) by an IgG-excess.

[0038] (17) Substances, acting anti-denaturing and partially re-naturingon biorelevant proteins, e.g. formamide, acetamide, anilide(fomlanilide, acetanilide), and their alkyl- and dialkyl derivatives,especially the non-toxic mono methyl-derivatives and otherwater-miscible compounds. They act by weakening the hydration and thedielectric constant (DK) of the medium. These substances can be combinedwith PEG, PVP and/or DMSO. The same substances and their combinationswith PEG, PVP, glycerol and/or DMSO are at the same time, according tothe invention, highly efficient cryoprotectants for cells and proteins.The PVP seems to be non-toxic, as it has been clinically used for yearsas plasma expander. Each class of the above described deblockingsubstances can also be used as additive to conventional vaccines.

[0039] (18) Anti-gamma-interferon, anti-M-CSF, anti-GM-CSF andanti-TNFalpha

[0040] (19) Liposomes, containing (a) cytotoxic agents (b) othercytotoxins, e.g. ricin, abrin

[0041] (20) Substances, preventing histamine release from mast cells,e.g. cromoglycin and intal

[0042] (21) Substances, reducing the Cl-concentration of theextracellular fluid (ECP)

[0043] (22) Substances, increasing-the ECF-concentration of HCO₃-ions.

[0044] According to the invention, a Ca-overload blocker, especiallycinnarizin (component I) should be combined with an antagonist ofβ-adrenoceptor, histamine-H₂-receptor and/or A₂-purinergic receptor,especially propranolol (component II). In addition, some selectedpreparations should be listed which can be used in combination with 1,2or more other substances (A+B, A+C . . . , B+C, B+D . . . , A+B+C, ABD .. . ). They have a special advantage to represent -as single substances-preparations which can be directly used clinically. They can be combined-as single or as complex preparations- with BRMs.

[0045] 1 Ca-antagonists (objective: prevention of the intracellularCa-over-load which results in the deblockade of guanylate cyclase and5-lipcxygenase and in the inhibition of CaM (calcium modulin)-mediatedactivation of Ca-ATPase and adenylate cyclase).

[0046] 1.1 based on nifedipin: adalate (26.071) or aprical 5/−10/-retard(26.072), or bayotensin/-mite (26.075)

[0047] 1.2 based on verapamil: azuparnil 40/−80/−120 (26.073) ordignover 40/−80 (2.081) or drostreakard 40/−80/−120 (26.083) orverapamil-ratiopharm (26.108)

[0048] 1.3 based on cinnarizin: cinnarizin-ratiopharm (36.035) orcinnarizin Siegfried (36.036) or cinnarizin R.A.N. 36.034) or cinnacet(36.633) or cerepae (36.032).

[0049] 2 β-blockers (Objective: inhibition of the cAMP-increasingsub-receptors for catecholamin, PGE1/PGE2 and histamine (H2-R)

[0050] 2.1 based on acetolol: neptal 400 (26.036) or prent 400 (26.040)

[0051] 2.2 based on metoprolol: lopresor/-mite (26.035)

[0052] 2.3 based on propranolol: indobloc 10/40/80 (26.032) or efectolol10/40/80 (26.024) or elbrol 40 (elbrol 80) 26.027)

[0053] 3 combination drugs: Ca-antagonists plus β-blockers: beloif(26.070) or tredalat (16.132) or nif-ten 50 (16.131)

[0054] 4 non-steroidal antiflogistics/antirhumatics (inhibitors ofcyclooxygenase/prostaglandin-synthetase)

[0055] 4.1 based on diclofenac: diclofenac-Wolff-25/50 (05.142) ordiclo-OPT 50, 100 retard (05.143) or diclophlogent (05.144)

[0056] 4.2 based on ibuprofen: dolgit 200/400/SL (05.149) or ibuphlogent200/−400 (05.161)

[0057] 4.3 based on indometacin: indo Tablinen (05.170) or indomatretard-rqatiopharm 75 (05.167) or amuno/retard (MSD) (05.129)

[0058] 4.4 based on ketoprofen: alrheumun (05.128)

[0059] 4.5 based on acetylsalicylic acid: spelt (05.122) or solpyron(05.121) or gepan/mite (05.117)

[0060] 5 drugs, affecting the intracellular pH_(i) (objective: impact onintracellular pH, K- and Na-ion, as well as on the HCO₃/CI⁻-ratio andherewith on the deblockade of hyper- or persistently activatedimmunocompetent cells, primarily macrophages) (a) hanooxygen (03.007) orgelum oral -rd (03.006(b)) acidovert (03.005) or acetolyt (03.001) ornephrotrans (03.004) or NaHCO₃ lg (03.003) (c) histinorm (05.210)

[0061] 6 drugs, correcting the redox-potential (NAD(P)H₂/NAD(P) andGSH/GSSG)

[0062] 6.1 drugs, improving the mercaptyl-/thionyl-,sulflhydryl-/disulfide-ratio (a) acetyl cysteine (23.119) oracetylcystein-ratiopharm 100/200 (23.120) (b) metalcaptase 150/−300(05.201) or trolovol (05.206)

[0063] 6.2 reducing drugs: cebion (83.099) or cedoxon Cassis (83.100) orcetebe (83.102) or resochin (05.203) or quensyl (05.202) or tauredon(10/20/50). The first two drugs are recommended ecially for their oraladministration.

[0064] 7 gold-based drugs (contribute -like chloroquine- to thestabilization of hyperactivated macrophages): ridaura (05.204) (oral) oraureostan 10/25/50/100 (05.199)

[0065] 9 methylxanthines (e.g. theophylline) (objective: stabilizationof the increased cGMP-level following the corrector of thecAMP/cGMP-ratio; methyl-xanthines inhibit not only thecAMP-phosphodiesterase but also the cGMP-PDE): theophyllinretard-ratiopharm 125/250/350/500 (27.082) or theospirex (27.084)

[0066] 10 antidiabetics (objective: mimicking of the insulin activity orantagonization of the glycolysis- and TCC-inhibiting andgluconeogenase-stimulating activity of glucagon)

[0067] 10.1 based on biguanidine (metformine): glucophage retard/-mite(11.070)

[0068] 10.2 based on tolbutamide: artosin 1.0 (11.037) or rastin-Hoechst(11.065) or tolbutamide 0,5 g/1 g (11.067)

[0069] 10.3 based on glibenclamide: azuglucon −3,5/−1,75 (11.038) ordiamicron (11.041) or glucononn 1,75/3,5 (11.051)

[0070] 10.4 based on glisoxepid: pro-diaban (11.064)

[0071] 11 guanylate cyclase (cGMP)-stimulating drugs

[0072] 11.1 based on isosorbid-dinitrate: coleb 20/−40 (54.044) ordignonitrat 40/−60/−100 (54.049)

[0073] 11.2 based on isosorbid-mononitrate: conpin 20/−40 (54.054) orcoragin 20/60 (54.046)

[0074] 11.3 based on glycerol-trinitrate: nitroglycerinretard-ratiopharm (54.023) . An alternative is the sydnonimin-derivativemolsidomine (e.g. molsidomine 1/2/4 from ct/Berlin). The workingmechanism on the cellular level corresponds to that of org. nitrates; Inboth cases, the activity of guanylate cyclase and herewith theintracellular cGMP-level is increased.

[0075] 12 inhibitors of xanthin-oxidase (XOD), with the aim to depressthe generation of oxygen radicals (see point (5)): allopurinol 300 Stada(43.007) or allopurinol Dorsch (43.008) or allopurinol-retard Woelm(43.011)

[0076] 13 some K-saving diuretics (due to K-retard-effect or indirectlydue to the stimulation of the H⁺/K⁺-antiport and herewith the pHi-rise).

[0077] 13.1 based on aldosteron-antagonist spironolacton:spironolacton-ratiopharm 50/−100 (02.016)

[0078] 13.2 based on triemteren: jatropur (35.060)

[0079] 13.3 based on amiloride: amiloride per se or in combination withhydrochlorothiazide as amilorid comp.-ratiopharm (35.063)

[0080] 14 carboanhydrase-blocker, e.g. acetazolamide (due to the impacton the pHi, on the glycolysis, gluconeogenesis and TCC/Krebs (citrate)cycle, as well as due to the depression of the glycolysis inhibitingFPK/fructose phosphate kinase): diamox retard (67.151) or diamox(39.006)

[0081] 15 oxygen carriers (objective: an improved O₂-transport to thehypoxic inflammatory tissue): oxoferin or TCDO (tetrachlorodekaoxid).The same or a better effect can be achieved with an oxygen therapy.Oxoferin and TCDO can be combined with ascorbate, succinate and/orfumarate.

[0082] 16 parathormone-antagonizing drugs (PTH acts via adenylatecyclase and cAMP immunosuppressive, like e.g. glucagon, histamine (viaH2-receptor), adenosine, PGE2, noradrenaline (via β1-adrenoceptor),adrenaline and isoproterenol (via β2-adrenoceptor). The macrophagesexpress receptors for (a) insulin (b) glucagon (c) histamine (d)serotonine (e) parathormone (f) calcitonine (g) somatotropine (h)somatostatine (i) PGE2 (j) cAMP (k) β-adrenoceptor (l) neuropeptides(endorphine) (m) arginin-vasopressin, and (n) transferrin.

[0083] 16.1 based on etidronacid :diphos (65.005)

[0084] 16.2 based on clodronacid: ostac (65.007)

[0085] 17 serene-compounds (objective: ROI-neutralization, stimulationof glutathione-peroxidase)

[0086] 17.1 based on Na-selenite-pentabydrat: selenase (GN-Pharm)

[0087] 17.2 based on ebselen

[0088] 18 Li-compounds (objective: increase of cytoplasmic pHi)

[0089] 18.1 based on Li-aspartate: Lithium-aspartat-Dragees 120 (70.235)

[0090] 18.2 based on Li-orotate: Lithium-orotat Tabl. (70.237)

[0091] 18.3 based on Li-carbonate: hypnorex retard (70.234)

[0092] 18.4 based on Li-sulfate: Lithium-duriles (70.236)

[0093] 19 some essential precursors, needed during switching fromcatabolic to anabolic function: 19.1. L-glutarnine 19.2. ribose 19.3.creatin(in) 19.4. ATP(ADP,AMP) and/or GTP (GDP, GMP, guanin) 19.5.unsaturated fatty acids+ascorbic acid 19.6. EPL 19.7. vanadin-compounds19.8. glutathion 19.9. folic acid

[0094] The best combinations are 1+2; 1 or 3+5; 1 or 3+6; 1 or 3+5+6 and5+6.

[0095] Especially recommendable are: in the group 1 1.3, in the group 5hanooxygen and in the group 6 6.1a and/or 6.2. These preferredcombinations can be further combined with 4, 12, 17 and/or 18, 7, 8, and10. An especially preferred combination comprises cinnarizine ascomponent I and propranol as component II. The conventional stimulatione.g. by BRMs can be supported by sub-dosed 11 and/or low-dosedlymphokines (e.g. IL-2, gamma IFN).

[0096] The fact that immunocytes react—like other somatic cells—ondifferent, Ca_(i) ²⁺-increasing signals has not been considered up tonow.

[0097] Therefore, the activation of immunocytes, as well as the insitu/in vivo deblockade of inactivated effector cells(monocytes/macrophages, Ts, NK-cells, K-cells) by alpha-sympathomimetics(e.g. phenylephrine), by Ca-agonists (e.g. Bay K 8644 or CGP 28392) andby β-receptor blockers (beta-blockers, β-sympatholytics) is recommendedaccording the invention. These, Ca_(i) ²⁺-increasing compounds can beused alone or combined with each other and/or with immunostimulators andBRMs. They are suitable for the in vitro and in vivo activation andreactivation/ deblockade of immunocompetent cells. β-blockers are ableto focus the activity of the physiological agonists noradrenaline(norepinephrine) and adrenaline (epinephrine) on the alpha1- andalpha2-adrenoceptor which favours cell activation (cGMP-rise andcAMP-drop).

[0098] According to the invention, a combination of agents with a doubleimpact on Ca_(i) ²⁺-level, i.e. by increasing Ca²⁺-influx and bymaintaining the so elevated Ca_(i) ²⁺-level in cytosol through the laterblockade of Ca-channels, is of a special interest. Such drugcombinations comprise on the one hand alpha-sympathomimetes, Ca-agonistsand β-blockers, and on the other hand Ca-channel blockers(Ca-antagonists, such as nifedipin, verapamil and diltiazem).

[0099] Examples of cGMP-increasing compounds are organic nitrites andnitrates, i.e. esters of the nitrous and nitric acid, such asamylnitrite, nitroglycerol, isosorbitnitrate and 5-isosorbitmononitrate,further Na-nitroprusside and parasympathomimetics; the latter can besubdivided in 3 groups: (a) choline ester (e.g. carbachol, bethanechol,metacholin); (b) alkaloids with parasympathomimetic activity (e.g.pilocarpin); (c) inhibitors of choline esterase (reversible inhibitors:physostigmin, neostigmine and pyridostigmin; irreversible inhibitors:fluostigmin and tetrastigmin).

[0100] In accordance with the further aspect of the invention, apreparation, consisting of (I) an agent, eliminating the hyperactivatedeffector cells, and (II) of alloreactive cells with preprogrammed celldeath, is recommended. The working mechanism of this preparation isbased on the replacement of “handicapped”, misprogrammed immunocompetentcells of the patient by the corresponding, in vitro pregenerated(specifically tailored) autologous or homologous/allogeneicimmunocompetent cells, a procedure called “microimmunosurgery”. This“microimmunosurgery” can be used in patients (a). with cancer(especially solid tumors) (b) with (retro)viral infections, includingAIDS, (c) with autoimmune disorders, and (d) with atherosclerosis-baseddisorders. In other terms, according to the invention, this preparationrepresents a combination (a) of breaking down (by impairing oreliminating) the resistance of the pathological set of patient'simmunocytes, and (b) of reinfusion of the ex vivo pregenerated(specifically tailored) immunocompetent cells. An additional effect isthe circumvention of the critical labile interphase by allogeneic and/orautologous immunocompetent cells, pretreated in vitro in a novel way.

[0101] The impairment or elimination of the “misprogrammed” (pathologic)immunocompetent cells of the recipient (patient) has been described inall details in the patent applications DE 3812605A1 and PCT/EP89/00403.Here, the novel in visor preparation of immunocompetent cells (a) forthe critical labile interphase, and (b) for the reinfusion of a new(healthy) set of specifically tailored immunocompetent cells, is dealtwith in detail. The problems, associated with bone marrowtransplantations (BMT) are an extreme susceptibility of patients forinfections and the unevitable “explosion” of residual tumor cells due tothe immunocompromized state of the patients. If the donor-bone marrowcells are not completely depleted of immunocompetent T cells, so calledgraft-versus-leukemia (GvL)-effect of the non-depleted donor T cellshelps to increase the resistance against the infection and the tumor;this advantage is, however, associated with the disadvantage of thegraft-versus-host (GvH)-reaction which shows similar fatalcomplications. With autologous BMT, this GvHR can be prevented. Abroader clinical use, e.g. in patients with solid tumors, is howeverdampened by the extreme immunologic lability of the BMT-conditionedpatients and the herewith associated enormous costs (ca. 160.000dollars/patient).

[0102] Both problems, the GvHR in allogeneic system and the extremeimmunological instability, associated with extreme costs, both inautologous and allogeneic BMT, can be solved by the 2^(nd) variant ofthe preparation according to the invention, implicating a novel in vitropremanipulation of allogeneic immunocompetent cells. In this way, boththe infection and the tolerancereinduction against the inevitableresidual tumor cells in the critical phase, following the removal ofprimary tumor can be prevented. In autologous BMT, the patient benefitsfrom the “inner immunological stability”, which minimizes the outer,extremely expensive sterility measures. This opens the ways to theintroduction of BMT in patients with solid tumors. A further advantageis the replacement of the patient-compromising whole-body-irradiationand/or high-dose chemotherapy by a selective depletion ofimmunocompetent T cells by different Mabs and Mab-basedimmunoconjugates. This improvement opens new ways for BMT, both inpatients with solid tumors and in those, suffering from autoimmunedisorders.

[0103] According to the invention, the preparation is suitable for atherapeutic procedure, consisting of 3 phases:

[0104] (a) In the phase I, the “misprogrammed” immunocompetent cells areeliminated by whole-body-irradiation, by high-dose chemotherapy, byspecific Mabs (or corresponding immunotoxins), directed against T cellsor their subpopulations (Ts in tumor patients, Tac-R⁺-cells inautoimmune disorders)

[0105] (b) In the phase II, the in vitro premanipulated, specificallytailored immunocompetent cells are injected into the patient, to conferon the patient immunocompetence in the critical, labile interphase.

[0106] (c) In the phase III, the patient is injected by ex vivopregenerated effector cells (CTLs, TILs, LAKs in the case of tumorpatients and autoantigen-specific Ts in patients with autoimmunedisorders).

[0107] Since the phase I is subject of the patent applicationPCT/EP89/00403 (author: P. Leskovar), only phase II and III will bedescribed here.

[0108] The effector cells, needed for the phase II are generated invitro as follows:

[0109] (a) Allogeneic (i.e. donor-) T cells/lymphocytes are culturedfirst in an isoleucine- or serum free medium to synchronize their cellcycle. Then, isoleucine or serum, respectively, is added and followingprogression in cell cycle (G₁. . . S . . . G₂-phase), the cells aretreated by mitomycin C. The mitomycin-concentration is adjusted in theway to allow a 2-5 cell division before cells die (e.g. 1-5 mg/10⁶cells). Instead of isoleucin or serum, other essential cell substratescan be used for the cell cycle arrest. Similarly, mitomycin C can bereplaced by different mitomycinC-homologs, such as BMV 25282 and BMY25067, as well as by other DNA-damaging, RNA-sparing substances, such asinhibitors of DNA-polymerase, DNA crosslinking cytotoxic agents andirradiation.

[0110] After 12-24 hrs, ideally 18 hrs of incubation with mitomycin C(or other compounds, damaging DNA in a reversible or irreversible way),the allogeneic (donor-)T cells are resuspended in a fresh medium.

[0111] To improve the in vitro activation, donor lymphocytes of the bonemarrow and/or peripheral blood can be preincubated in a kind ofone-way-MLC/MLR with the recipient lymphocytes (favorably withT-depleted or preselected MHC II-positive B cells and/or adherentcells).

[0112] This preincubation occurs in isoleucin- or serum free medium; therecipient MHC II-positive cells (B cells) have to be, however,pretreated by mitomycin C or irradiation so that they stay metabolicallyactive but unable to proliferate. During the later mitomycin C-treatmentof allogeneic (donor-)lymphocytes (T cells), they get an additional doseof cytotoxic agent, leading to their selective death. The sopremanipulated donor cells are activated during the contact withpatient's MHC II-positive cells (B cells, monocytes/macrophages,activated T cells), following their infusion into the patient; theysecrete IL2 and other cytokines which are reduced or absent inimmunoincompetent recipients (due to the lack of mature helper T cells.)

[0113] These donor cells can, however, not induce the fatal GvHR orGvHD, as they are “preprogrammed” and die after few cell divisions.

[0114] (b) By an alternative procedure donor's mitomycin C-pretreatedPBMs(without bone marrow) are injected in the first phase, and donor'sT-depleted bone marrow is post-transfused in the second phase. The donorT cells, pretreated in this way, can be replaced or combined withallogeneic (donor-) LAK cells.

[0115] Normally, only autologous LAK cells are used therapeutically.

[0116] According to the invention, allogeneic (donor-)instead of or inaddition to autologous LAK cells should be used therapeutically. TheGvHD-complication is not a problem, because the LAK cells consist of upto 90% activated NK cells and of up to 10% non-MHC-restricted CD3+(T)cells. The T subpopulations which are responsible for the alloreactionand GVHD, cannot survive during the LAK-generation in vitro, due to thelack of antigen (i.e. alloantigen of recipient MHC II-positive cells). Tcells can, however, be depleted in vitro by specific Mabs orimmunotoxins for reasons of an additional security. Alternatively, L AICcells can be pretreated in vitro by mitomycin C (or other DNA-damagingsubstances), as described above.

[0117] (c) If for unknown reasons GvHR or GvHD are observed, so a novelstrategy can prevent these complications and the GvHD-establishment ingeneral, according to the invention. The principle is the in vitrogeneration of alloreactive T cells (a) from recipient or (b) from athird person (second donor), directed against donor cells by means ofthe MLC/MLR-technique.

[0118] The donor (first donor) cells have to be pretreated byproliferation-preventing mitomycin C-doses in order to be able to act asstimulator cells in the MLC.

[0119] The responder cells (from recipient or 2^(nd) donor) arethereafter treated by mitomycin C (or other DNA-damaging substances) ina way, allowing the cells to divide for 2-5 times before they die.

[0120] To increase further the efficiency, the autoaggressivesubpopulation of patient T cells can be preeliminated by immunotoxins,consisting of cytotoxin (e.g. abrin, ricin, doxorubicin,¹³¹I-radionuclid) plus IL-2 or anti-Tac/II-2-R-Mab.

[0121] According to the invention, the so pretreated effector cells canbe frozen, similar to the effector cells with restricted lifespan,described under (a),(b) and (c), in a medium, containing 8-15% DMSO andPVP or PEG of different concentration and mol weight (patent applicationDE 3812605 A1 and PCT/EP89/00403).

[0122] The addition of PVP and/or PEG improves the viability andpreserves-in contrast to the sole DMSO-addition-the preactivated stateof cryopreserved effector cells. In this way, a repeated infusion ofeffector cells into the recipient became possible.

[0123] An additional improvement is the separation of donor adherentcells (macrophages/monocytes) before the mitomycin C-treatment, followedby their later readdition to the mitomycin C-pretreated effector cells(T cells, lymphocytes) and infusion into the patient.

[0124] Mitomycin C can be combined with interferon (alpha, beta, gamma),TNFalpha, DMSO, vitamin A and E, as well as the (re)differentiationsubstances, such as butyrate.

[0125] The following procedure is also especially preferred: Patient'slymphocytes can be clonally expanded in vitro into tumoricidal CTLs andplasma cells by mitogenic lectins (PHA,ConA) and mitogenic antibodies(anti-CD3/Ti, anti-CD2/T11). This in vitro postexpansion comprises onlythe blastogenically pretransformed T and B clones, i.e. memory T and Bcells. Since tumor patients have a persistent contact with tumor cellsand AIDS/ARC/LAS-patients with HIV and opportunistic infections,respectively, their blood contains corresponding memory cells. Dependingon the cell structure conditions, these memory cells can be directedtoward CTLs (CD8⁺, and CD4⁺) or Ts cells; this opens new ways for theirtherapeutic use (a) in tumor- and AIDS-patients, and (b) in patientswith autoimmune disorders.

[0126] If the resistance (immunocompetence) of the patient istemporarily down-regulated, the infusion of in vitro clonallypostexpanded patient's lymphocytes (T cells) can have an essentialimpact on the disease development. The expansion of CTLs can be achievede.g., by the PHA-treatment for 3-6 days. The autotolerant Ts cells,necessary for the therapy of autoimmune disorders, can be generated e.g.by the treatment with PWM for 7 days or with the PHA for 3-4 weeks. TheCD8+-rise is accompanied by a CD4⁺-drop. These Ts cells can be culturedfor more than 6 months under following conditions: 2 times weekly, IL2is added to the medium and cells are restimulated by feeder cells andPHA every 2 weeks. These cells suppress the proliferation of autologousand heterologous CD4⁺T cells when stimulated by PWM, OKT3 ortetanus-toxoid.

[0127] Alternatively, Ts cells can be generated in vitro in the presenceof PGE2, anti-IL1, anti-IL2, anti-IL4, cyclosporinA, rapamycin and/orFK506.

[0128] The Ts-depleted cytotoxic effector cells, needed in the tumor andAIDS/ARC-therapy, can alternatively be enriched by the elimination ofCD8⁺cells (by means of Mabs or immunotoxins). The spared CD4⁺T cells areable to induce in vivo new CTLS; this process is accelerated byanti-PGE2, anti-lipocortin/macrocortin and anti-TGF-beta. InAIDS/ARC/LAS-patients, it is advantageous to preimmunize the patientwith the lysate of opportunistic infections, before the in vitroexpansion of memory cells; alternatively, a healthy donor can bepreimmunized (a) ,with a viral antigen (8p 120) and (b) with mentionedlysate.

[0129] The so generated clonally postexpanded allogeneic memory cells (Tand B cells) can be treated by mitomycin C, washed and injected into theAIDS-patient.

[0130] Atherosclerosis and other coronary diseases show an autoimmunegenesis (our own experiments, reports of others, e.g. W. Hollander);therefore, the agent(s), based on “microimmunosurgery”, are able toreplace or support the “conventional” treatment.

[0131] According to the invention, hyperactivated B or T or both B and Tcells of atherosclerotic patients are depleted/inactivated by means of“microimmunosurgery”. It is advantageous to replace them by the in vitropregenerated Ts fraction. These Ts cells can be generated by a simple,3-5 week incubation of patient's PBMs with ConA or PHA. Alternatively orsupportingly, the patient can be treated by special preparations whichinactivate the hyperactivated macrophages; they consist ofFab/F(ab′)₂-subunits (labeling) or immunotoxins (depletion), directed(a) against the (acid-labile and acid-resistant) Fc(gaTnma)-receptor,(b) against the complement receptor (CRT, CR3), (c) against thescavenger/AcLDL-receptor (I and II), (d) against thegamma-interferon-receptor and /or (e) against theLPs/endotoxin-receptor. Alternatively, the biotechnologicallysynthesized receptor per se or its subunits can be used therapeutically.

[0132] In addition, denatured (e.g. heat-denatured) complement subunits(clq, c3b, c3d etc.) or biotechnologically produced defect C-componentscan be used; they can be combined with antioxidants (vitamin E,A,probucol). The in vivo neutralization of the solubilized ApoB,E- andApoE-receptor in the patient's plasma, following their quantitativedetermination in vitro, is also advantageous.

[0133] An alternative way comprises a combination (a) withCa-antagonists/Ca-channel blockers (verapamil, nifedipin, dilthiatem),(b) with cGMP-increasing substances (e.g. Na-nitroprusside, org.nitrates), and (c) with pHi-raising substances. In this way, thereplacement of “wrongly programmed” (hyperactivated) macrophages by“fresh” monocytes should take place.

[0134] The protracted or repeated microimmunosurgery includes theprevention of neutralizing antibodies against xenogeneic (mostly murine)Mabs and Mab-based immunoconjugates by substances, specified in thepatent application PCT/EP89/00403, as well as by some procedures,described here:

[0135] (1) Prevention of neutralizing antibodies by the pretreatment ofrecipient with Mabs which are coupled as a kind of hapten to a tolerogenas carrier. Examples of such tolerogenic carriers are polyethyleneglycol(PEG), polyvinylpyrrolidone (PVP) and different copolymers of D-aminoacids (e.g. D-glutamine-lysine, shortly D-GL).

[0136] (2) The same principle (like under (1)) can be applied todifferent immunoconjugates, e.g. conjugates of Mabs (directed againsttumor cells, viral/bacterial infections, leukocytesubpopulations) with(a) cytotoxins, such as ricin or abrin, (b) with cytotoxic agents, suchas doxorubicin, (c) with radionuclids, such as ¹³¹I, and (d) with targetcells-starving enzymes, such as arginase, asparaginase etc. Atwo-step-administration (first sub-immunogenic, then immunogenic dose),can additionally potentiate the effect.

[0137] (3) Neutralizing antibodies against xenogeneic (murine) proteins(e.g.Mabs) can be prevented also by strictly monomeric,molecular-dispersed structure of these xenogeneic proteins, which can beachieved by their pretreatment (a) by mercaptoethanol (b) by glutathione(c) by N-acetyl-cysteine (d) by penicillamine D (e) by other substanceswhich support the disulfide-to-thioVsulfhydril-interconversion, as wellas by 6M-urea and guanidine-hydrochloride.

[0138] (4) Prevention of neutralizing antibodies by a direct in vivo useof Mab-producing plasma cells, which induce a low-zone tolerance againstxenogeneic proteins by secreting strictly monomeric Mabs; these are notxenogenized by in vitro manipulation.

[0139] (5) Prevention of neutralizing antibodies by the direct in vivouse of Mab-secreting hybridoma cells, which were pretreated in vitro bymitomycin C and/or other DNA-crosslinking cytotoxic agents (restrictedlifespan, 2-5 cell divisions only).

[0140] (6) Prevention of neutralizing antibodies by aggregate-preventingsubstances, e.g. protein-solubilizing tensids (e.g. salts of higherfatty acids, Iysolecithin) at extremely low concentrations).

[0141] As next, some further details to this invention will be dealtwith:

[0142] The hyperactivated, suppressive macrophages can be inactivated asfollows:

[0143] (a) by antioxydants (b) by inhibitors of enzymes, involved inROI-synthesis (c) by PAF-blockers (d) by PLAZ/PLC-inhibitors and/or (e)by Ca-channel blockers/ Ca-antagonists. In the 2^(nd) phase, therecruitment of new macrophages from monocytes is foreseen.

[0144] The in vitro generation of blastogenically pretransformed(memory) cells can be accelerated if patient's PBMs are first treated byanti-CD8- and/or anti-CD3-Mabs plus complement or by correspondingimmunotoxins. This leads to a partial Ts-depletion and should befollowed by the treatment with mitogens (lectins or mitogenic Mabs).

[0145] In patients with autoimmune disorders, the addition of Ts-stimulating substances (anti-HLA-DR-Mab, anti -LFA- lbeta-Mab,cyclosporinA, corticosteroids, FK 506, rapamycin, ConA) and in patients,suffering of cancer or AIDS (and other viral diseases), the addition ofTc(CTL)-promoting substances (anti-HLA-DQ-Mab, anti-LFA-alpha-Mab,cyclooxygenase-inhibitors such as aspirin, indomethacin, anti-PGE-Mabetc.) is recommended. The preferred generation of Tc instead of Ts cellscan be observed in vitro also when adherent cells are removed. Thistechnique is based on the effect of ex vivo specifically tailoredeffector cells on the disease regression; the patient's immunologicresistance has to be temporarily reduced by anti-CD8-Mab and/oranti-CD3-Mab (in the case of tumor- and AIDS-patients) and byanti-CD3-Mab (in patients with autoimmune disorders): The therapeuticefficiency in tumor patients is further increased if patient's tumorcells are incubated in the presence of gamma-interferon, TNFalpha and/or5-HETE in order to induce MHC I and/or MHC II-postexpression on thecells and are then reinjected along with patient's specificallytailored, in vitro postexpanded memory cells.

[0146] A further increase in efficiency can be achieved by the confusionof patient's glutaraldehyde-pretreated macrophages which have beenpreincubated with tumor antigen. An additional advantage is theconfusion of patient's inactivated (preirradiated orglutaraldehyde-pretreated) leukocytes (PBMs) which results in theinduction of antiidiotypes, directed against patient's Ts cells. Theinfusion of hybridoma cells, based on patient's tumor cells and MHCII-positive autologous and homologous cells is advantageous, as well.

[0147] The above discussed LAK/TIL-techniques can be further improved byadding mitomycin C-pretreated allogeneic (donor) T cells to LAK or TILcells. This increases the number of lymphokine-secreting cells which isespecially important for the TIL-technique.

[0148] These premanipulated allogeneic cells are able to play a similarpositive role in bone marrow recipients. The preexpanded allogeneiccells can be used also in the treatment of autoimmune disorders andGvHD, if they are pretreated by DNA-damaging agents. The RES-elimination(99%) of LAK and TIL cells due to their “xenogenization” during the exvivo manipulation can be reduced, according to the invention, byaddition of alpha2-macroglobulin, antitrypsin and/or cortisone to themedium.

[0149] A variant of the described procedure renders the externallycontrolled in vivo production (a) of cytokines (e.g. TNFalpha, IL-1,IL-2, IL4, IL-6, IL-3, G-CSF, M-CSF, GM-CSF), (b) of hormones (e.g.insulin, parathormone etc.), and (c) of other physiologically importantcell factors possible. The principle is the transfection of donorspecific recipient T cells by corresponding, these cytokines or hormonesencoding genes; the preselection of alloreactive, donor-specificrecipient T cells can be achieved by a (repeated) one-way- MLC(stimulator cells: donor-PBMs; responder cells: recipient-PBMs).

[0150] After the reinfusion of the transfected recipient T cells, thesecan be repeatedly reactivated in vivo to secrete cytokines or hormonesby injecting i.v. inactivated (mitomycin C-pretreated or preirradiated)donor-PBMs. After the in vitro pregeneration of autologous, alloreactiveT memory cells, directed against donor A, donor B, donor C etc.,different functions can be transfected donor-dependently into theserecipient memory cells and “recalled” in vivo, following the reinjectionof these manipulated autologous cells into the recipient. By theintratumoral injection of these cells, the effect can be localized tothe tumor tissue. A similar in vivo “switching on” of the desiredfunction can be achieved by the following procedure: the recipient Tcells are first primed in vivo by a model antigen and then clonallypostexpanded in vitro in the presence of the same antigen. In the nextstep, these cells are transfected by the gene of interest, e.g.cytokine- or hormone encoding gene(s) and reinjected into the recipient,favorably after the temporary depletion of recipient's immunocompetentcells. Later restimulation of the recipient by the same model antigen“turns on” the desired (“transfected”) cell function. Representatives ofsuch antigens are tuberculin and other antigens, used in cutaneoustests, as well as haptens (DNCB, DNBB, DNBS, TNBS etc.).

[0151] Instead of the above described alloantigens and model antigens,low-dose allergens can be used.

[0152] This externally controlled in vivo secretion of cytokines is ofspecial interest in the therapy of strongly immunocompromised patients(AIDS/ARC/LAS-patients, patients with advanced cancer, recipients ofbone marrow grafts).

[0153] The above discussed transplantation (a) of organs (b) of bonemarrow and (c) of mitomycin C-pretreated, hormones (e.g. insulin) andcytokines-secreting allogeneic or xenogeneic cells and hybridomas can beessentially improved by the following procedure: First, the immuneresistance of the recipient must be temporarily down-regulated(“broken”) by specific antibodies (anti-panT- or anti-CD8-Mabs) orMab-based immunotoxins. Then, the in vitro pregenerated suppressor Tcells (Ts)(see above!) are reinjected, immediately before thetransplantation. The main advantage over the “conventional” grafts isthat here—in contrast to the “conventional” grafts—the recipient is notconfronted “unexperienced” with the MHC II-positive donor cells (bonemarrow macrophages and B cells, as well as “passenger lymphocytes” inorgan grafts) but preinjected by allotolerant Ts cells; thesedonor-specific Ts-memory cells direct the CD4/CD8-double positiveprecursor cells (inducer/transducer suppressor cells) towards theTs-effector cells before the alloreactive Tc/CTL cells predominate. Thisseemingly minimal deviation in the procedure can decide about thesurvival of organ- and bone marrow recipients.

[0154] The tolerance against the organ or bone marrow graft, as well asagainst the hormone (e.g. insulin) or cytokine-producing allogeneiccells can be induced also in the following way: Alloreactivedonor-specific recipient T cells are selected and expanded in vitro bymeans of MLC (responder cells: recipient PBMs, stimulator cells: donorPBMs). The repeated MLC results in a 95% enrichment of thesealloreactive T cells. The next step is the inactivation of recipient'sdonor-specific alloreactive PBMs or T cells by irradiation or mitomycinC: Before they are injected into the recipient, the latter must betemporarily rendered immunoincompetent (by anti-CD3- or anti-CD1- oranti-CD8-Mab). The principle here is the induction of anti-idiotypes inthe recipient (in vivo), before this encounters MHC II-positive donorcells (bone marrow cells or “passenger lymphocytes”). Therefore, thisinduction of anti-idiotypes has to be carried out several days beforethe real transplantation.

[0155] Problems, associated with the transplantation, such as immunesuppression or susceptibility for infection can be reduced by theco-infusion of in vitro inactivated allogeneic (donor) MHC II-positivecells (B cells, macrophages), along with the allotolerant Ts cells.These MHC II-positive cells can be pregenerated in vitro by a kind ofMLC (responder cells: recipient-PBMs, stimulator cells: donor-PBMs),frozen and during or after the transplantation repeatedly reinjectedinto the recipient.

[0156] The precursor cells can be directed toward Ts cells by theaddition of anti-HLA-DR- and/or anti-LFA-lbeta-Mabs. In contrast, thegeneration of Tc/CTLs can be induced in the presence of anti-HLA-DQ-and/or anti-LFA-lalpha-Mabs. The rise of MHC II-positive APCs(“passenger lymphocytes”), which is critical for the graft failure, canbe prevented by the addition of anti-HLA-DR-Mab or the correspondingFab/F(ab′)₂-subunit and/or Ca-channel blocker (verapamil,nifedipin,dilthiazem).

[0157] A further improvement of the above discussed LAK/TIL-techniquecan be-achieved (a) by co-infusion of mitomycin C- preinactivatedallogeneic, MHC II-positive cells (B cells, adherent cells), and (b) bythe addition of sub-dosed corticosteroids and/or serum proteinaseinhibitors (alpha2-macroglobulin or alphal-antitrypsin) to the mediumduring LAK or TIL generation. The so modified culture medium preventsthe in vitro xenogenization of LAK and TIL cells and herewith theirearly RES-elimination in vivo.

[0158] The activation of tumoricidal/virucidal effector cells can beachieved also through a controlled treatment of these effector cells (NKcells, T cells, macrophages) (a) by fusogenic substances insub-fusogenic concentrations (e.g. PEG, PVP), further (b) byelectrofusion under sub-fusogenic conditions (1000-5000 kHz; 10-150V/cm²) and/or (c) by proteolytic enzymes and lipases.

[0159] The above described hybridomas can be used, according to theinvention, also to stabilize and to establish herewith new cell lineswhich normally wouldn't survive in vitro.

[0160] An improved localization of the above described, transfected,cytokines or hormones secreting T cells can be achieved by “arming” ofthese transfected cells with bifunctional Mabs which recognize theseboth transfected cells and the tumor cells.

[0161] In order to induce a kind of allergic reaction against tumorcells, the “conventional” anti-tumor-Mabs which are normally of theIgG-isotype, can be combined with anti-tumor-Mabs of the IgE-isotype.The latter can be produced in vitro by the “isotype switching” of plasmacells from the IgG- to the IgE-production; the process of “isotypeswitching” can be induced by addition of anti-gamma interferon andanti-IL2-Mab, as well as IL3, IL4 and IL5 to the medium. A simultaneousaddition of anti-CD8-Mab is advantageous. In the case of anti-tumor-IgGsthe immortalization (hybridoma formation) is carried out before and inthe case of anti-tumor-IgEs after the “isotype switching” of plasmacells. Alternatively, anti-tumor-Mabs of the IgE-isotype can beconstructed by the conjugation of xenogeneic (murine) anti-tumor-Mabs(more precisely: Fab/F(ab′)₂-subunits) with human Fc-subunits.

[0162] According to a further variant, a direct in vivo use of hybridomacells, produced before (IgG) or after (IgE) the “isotype switching” isrecommended; these hybridoma cells have to be pretreated byDNA-damaging, RNA-sparing agents (e.g. MMS, mitomycin C etc.)

[0163] In a further variant, anti-gamma-interferon plus IL4 (IL-3, IL-5)are directly injected into the patient; herewith the activity of T_(H2)cells is increased and that of T_(H1) cells depressed which leads to anearly “isotype-switching”.

[0164] The next strategy is the replacement of the immortalizing(transformed) partner cell (e.g. NS-1) in the hybridoma and quadromacell by MHC II/HLA-DR-positive (allogeneic) cell. In this way, thehybridoma and quadroma cells can be externally switched on and off. Thenew procedure is based (a) on the in vitro pregeneration of alloreactiveT cells of the recipient, directed against the donor A and furtherdonors (donor B. donor C etc.) by means of the (repeated) MLC, (b) onthe fusion of preselected alloreactive memory-T-cells with partner cellswhich are used for “conventiona” hybridizations (e.g. anti-tumor-Mabproducing plasma cells), as well as (c) on the reinfusion of the sogenerated hybridoma cells into the temporarily immunocompetentrecipient. These hybridoma cells which are well tolerated, as they arefully (in the case of human autologous plasma cells) or partiallyautologous (in the case of murine plasma cells), can be laterreactivated repeatedly by the injection of donor-lymphocytes.

[0165] A kind of tumor-directed “autoreactivity” can be achieved asfollows: tumor cells of the patient are stimulated in vitro bygamma-interferon (TNFalpha, 5HETE) to post-express MHC II (and MHC I) onthe cell surface. Alternatively, tumor cells can be fused (a) withautologous or (b) allogeneic, MHC II-positive cells and washedthoroughly.

[0166] A further xenogenization of patient's tumor cells can be achievedby the fusion with LPS-containing gram-negative bacteria and/or yeastcells. After their inactivation, these manipulated tumor cells areinjected into the patient who has to be rendered immunoincompetenttemporarily (by injection of anti-CD3-, anti-CD1, anti-CD2- oranti-CD8-Mab). In this way, the autoaggression against tumor cells canbe induced.

[0167] Hybridoma cells, arising from patient's tumor cells and MHCII-positive allogeneic cells (e.g. allogeneic B cells) are able toaccelerate the induction of the autoaggressive reaction against tumorcells if they are combined with (repeated) injection of inactivated PBMsfrom that donor whose MHC II-positive cells were used as partner cellsfor the hybridization with patient's tumor cells (see above).

[0168] The same techniques can also be used for the improvement ofconventional vaccines (e.g. against bacterial and (retro)viralinfections, including HIV). So, the efficiency of conventional vaccinesis essentially increased if they are combined with anti-CD8-Mabs (or thecorresponding immunotoxins) and/or with complete and incomplete(Freud)-adjuvant. It is also advantageous to combine the 1^(st)vaccination (“priming”) but not the 2nd vaccination (“boosting”) withanti-B-cell-Mabs (e.g. anti-CD19-, anti-20-, anti-CD21-, anti-CD22-Mab).The in vitro preformed immune complexes, composed of pathogen andanti-pathogen (IgM-isotype) with or without bound complement orcomplement-subunits (clq, c3b/c3d etc) can also be promising.Alternatively, the efficacy of the vaccine can be improved byIgE-inducing IL3, IL4, IL5 and anti-gamma-interferon.

[0169] The above discussed GvHD can be prevented also by autologous PBMswhich have been also preactivated by a repeated MLC. In this MLC, theresponder cells are autologous (recipient) PBMs and stimulator cells theallogeneic (donor) PBMs; the latter have to be preirradiated ormitomycin C-pretreated. The repeated co-incubation of donor- andrecipient-PBMs leads to the generation of highly efficient alloreactivememory cells which are able to eliminate the GvHD-causing donor-T-cells.

[0170] The autologous BMT can be improved: (a) by preelimination ofsuppressor-T-cells in vitro, e.g. by anti-CD8-Mabs or correspondingimmunotoxins, (b) by in vitro preactivation of effector cells, (c) byaddition of autologous and/or allogeneic LAK-cells (the allogeneicLAK-cells can, but need not be predepleted of CD3-positive cells), (d)by addition of autologous, CD8⁺-depleted PBMs to the autologous bonemarrow cells (the PBMs should be preactivated in a tumor-specific way),as well as (e) by the addition of allogeneic, favorably preactivatedallogeneic PBMs which have to be pretreated by mitomycin C or otherDNA-damaging and RNA-sparing substances in order to confer to thealloreactive subpopulation a restricted lifespan.

[0171] According to the invention, the stepwise addition of increasingdoses of anti-CD8- or anti-CD3-Mab plus complement (or of correspondingimmunotoxins) to the patient's PBMs allows the determination of thecritical T cell number/ml which directs the immune response fromsuppression to stimulation (deblockade) of T4 and B memory cells. The sopremanipulated autologous PBMs mimic the favorable clinical situation,when autologous marrow cells are collected during remission (PR or CR)and not during relapse.

[0172] This technique is recommended especially for patients with solidtumors, as well as for AIDS/ARC/LAS-patients.

[0173] The above mentioned LAK dells show the special advantage thatthey cannot be contaminated with (tumor-specific) Ts cells; a furtheradvantage is the lymphokine-production by autologous T cells, directedagainst allogeneic LAK-cells, which is especially important forimmunocompromized recipients. The LAK-cells of one or more persons couldbe frozen and used for different patients, especially after ananti-CD3-pretreatment (“standardized LAK-cells”).

[0174] As next, some additional improvements of the techniques describedabove should be quoted: So, the restricted lifespan of hybridoma cellscan be achieved by the change of the ratio of partner cells duringhybridization, instead of the treatment of hybridoma cells withDNA-damaging and RNA-sparing agents. it concerns the chemical (PEG) andviral (SV-40) fusion, as well as the electrofusion.

[0175] If the ratio of partner cells which contribute to theimmortalization of hybridoma cells in comparison to those whichcontribute the desired function is reduced from “classical” 1:1 to 1:2;1:3; 1:4; 1:51: . . . 1:10, a controlled longevity of hybridomaconstructs can be achieved, similar to that, achievable by low-dosemitomycin C. By an early fusion of instable cell lines, which don't growex vivo, with transformed cells, these cells can be “stabilized” and areable to survive in vitro.

[0176] According to the invention, the fusion of lymphokine andhormone-producing cells with transformed cells, followed by themitomycin C-treatment, opens for the first time the possibility of adirect in vivo implantation of cytokine and hormone-producing cells(e.g. insulin production). The so pretreated secretory cells die after alimited number of cell divisions and can be substituted by a new set ofpremanipulated cells; this allows an efficient control of the cytokineand hormone level.

[0177] The tolerance against such secretory cells can be achieved (a) bythe simultaneous treatment with immunosuppressive substances (cytotoxicagents, sub-dosed anti-CD4- or anti-MHC II-Mab), (b) by cellimplantation in thymus and/or (c) by “autologization”, i.e. by use ofautologous partner cells for hybridization. Further comments: InAIDS-patients, the in vitro presensibilization of patient's lymphocytes(T cells) by preirradiated of mitomycin C-pretreated donor-lymphocytes(T cells), followed by the transfection of alloactivated patient's Tcells with cytokine or anti-HIV-Ab-producing genes, and reinfusion ofthe so transfected autologous T cells into the patient is recommended. Apart of transfected cells can carry the gene for the IgG-isotype and theother part that for the IgE-isotype. The later “switching on” of thecytokine and antibody-production occurs by the injection of inactivateddonor lymphocytes.

[0178] According to a simplified version, the patient's T cells ingeneral, not only his donor-specific alloreactive T-subclones can betransfected in vitro with cytokine and/or anti-HIV-antibodies-encodinggenes, and reinjected into-the patient. Again, the later “switching on”of the in vivo cytokine and antibody production occurs by the injectionof donor-lymphocytes; the system works because patient's lymphocytesalways contain T cells which are alloreactive against the donor.

[0179] In conventional vaccines, the ratio between “positive” (ThandTc/CTL) and “negative” (Ts) memory cells can be increased as follows:(a) In the 2^(nd) phase, i.e. 4-6 days after the vaccination, thecouterregulation of persistently activated macrophages can be delayed byCa-channel blockers and macrophages-inactivating measures(ROI-inactivating agents, such as retinol, tocopherol or carotinoids,glutathione, ascorbate, radical-scavengers). (b) With anti-HLA-DQ (notDR) and anti-LFA-lalpha (not beta), the (Th+Tc)/Ts-ratio can bepositively influenced. (c) A similar effect (increase of the(Th+Tc)/Ts-ratio) can be achieved byantilipomodulin/anti-lipocortin/anti-macrocortin-Mabs and/or mellitin.(d) Ba the conjugation of pathogen antigens (viral or bacterialstructures) to carriers which are used as components of common vaccines,such as tetanus and diphterytoxoids, tuberculin, inactivated M.tuberculosis and BCG; the efficiency of conventional vaccines can befurther increased. (e) an additional improvement is the use ofanti-thymosine-alpha7 (not alphal). (f) The efficiency of vaccines canbe also increased by anti-PGE2(El) and by PGF2alpha. (g) The resistanceagainst pathogenic microbes can be increased as well, if first contactwith antigen structures of the pathogen (e.g. gp 120/160 or gp 41 ofHIV) occurs via alveolar macrophages which prevents its early contactwith Ts cells (as in the case of the i.v. administration). Therefore,the sensibilization by aerosol-spray as the only or at least first step(“priming”) is recommended. (h) In the 2^(nd) phase of vaccination, i.e.4-7 days after vaccination, the Ts generation should be prevented byanti-PGE2- and anti-interferon-Mabs. (i) The in vitro preformedIgE.antigen:IgE-complexes are able to activate the IgE-R-expressingcells (macrophages, NCF-A-, ECF-A- and kininprotease-secreting mastcells) in the critical early phase.

[0180] Some additional techniques: (a) Tumor patients can be treated byinactivated hybridoma cells, arising from patient's tumor cells andallogeneic (donor) cells of the same origin, i.e. the same organ liketumor. An example are leukemia cells, fusioned by the samelymphocyte-subclass of the healthy donor. (b) The Th(=T_(M)) cells canbe stimulated in vivo if the injection of antibodies of the IgM-isotypeis followed by the injection of the corresponding antigen. Analogously,the TS(T_(G)) cells can be stimulated in vivo by the successive infusionof IgG and the corresponding antigen. (c) Similarly to the abovedescribed “allogeneic switching”, autologous lymphocytes can be primedin vitro by a model antigen and the resulting memory cells transfectedby cytokine and Mab-encoding genes. After the reinfusion they respond tothe resensibilization by the same model antigen. The strongest responseshow the cutaneous antigens. It is advantageous to use the preexistentmemory cells which stem e.g. from previous vaccinations (e.g.tuberculin) or sensibilization (DNCB, DNBB, TNBS) . (d) By anti-CD3-,anti-TCR/Ti-, anti-CD2/T11- and other mitogenic Mabs, the memory but notnaive T cells can be stimulated to proliferate; in naive/non-primed Tcells, the mean distance between membrane antigens of the samestructure, e.g. CD3 or Ti is too long to be “bridged” by bothFab-subunits. Therefore, novel antibodies are recommended according tothe invention, containing two or more anti-CD3(Ti, CS2. . . )-Mabs ortheir Fab/F(ab′)₂-subunits, conjugated to different spacer-molecules;this renders a direct crosslinking of CD3-, Ti-, CD2- and other membranestructures possible even on naive Th- cells. In this way, anAPC-independent activation of naive Th-cells is possible which can be ofrelevance in immunosuppressed AIDS and tumor patients.

[0181] In order to prevent at the same time the neutralizing antibodiesagainst xenogeneic (murine) Mabs, the use of strong tolerogens,especially polymers/copolymers of D-amino acids, e.g. D-GL, or PEG orheparin or sialic acid-based polymers, is recommended.

[0182] The efficacy of the novel type of antibodies can be furtherincreased by their combination with interferon which increases thesurface density of structures to be crosslinked.

[0183] The tumoricidal and/or virucidal effector cells can be activatedin a novel way by conjugating Mabs or their Fab/F(ab′)₂-subunits,recognizing (a) cytokine receptors or (b) growth factors (EGF, IGF, PDGFetc.) with spacer molecules, e.g. the CH₂)n-chain or D-GL. D-polylysineas spacer molecule is of special interest, as it is a tolerogen and asit facilitates—through the positive charge—the approach of theMab-construct to the target cell. The same is true with the polymers orcopolymers of other basic, D-aminoacids. The here described newprinciple of introducing spacer molecules as carriers of Mabs with thesame or different specifity, is also applicable for all simple orcombined Mabs, specified in the patent applications PCT/EP ()/00403 andDE 3812605A1. (e) Since LAK cells consist to 90% of NK cells and to 10%of non-MHC-restricted CD3⁺-cells and are free of alloreactive,GvH-inducing T cells, the allogeneic LAK cells can be used to circumventthe immunoincompetent phase which is critical for later relapses (a) inbone marrow recipients and (b) in patients, immediately following tumorsurgery. LAK cells can be depleted of T cells before their infusion intothe patient. LAK cells can be frozen by a special procedure (withPEG/PVP-addition to DMSO), preserving their preactivated state.

[0184] *(f) Atherosclerosis seems to be induced by an autoimmune primer,including hyperactivated macrophages. The latter can be depressed (a) bycorticosteroids (b) by Ca-antagonists (c) by masking the Fc andCR1/CR3-receptors with Fab/F(ab′)₂-subunits of the anti-receptor-Mabs(d) by ROI-reducing agents and radical-scavengers, and (e) byanti-CD19(CD20, CD21, CS22)-Mabs (temporary depletion of CIS-producingB-cells). Within the inflammatory tissue, the immunocytes are exposed(a) to hypoxic/anoxic and/or (b) acidic micromilieu. (a) The unfavorableredox-potential in this tissue has to be normalized by O₂-carriers (e.g.oxoferin, TCDO=tetrachlorodekaoxid) and/or by other electrone acceptors(e.g. ascorbate, dehydro-ascorbate, (poly)unsaturated fatty acids). (b)The low pH in the inflammation tissue inhibits immunocompetent cells. Bythe pH-rise, the H⁺/Na⁺-pump of immunocytes must be relieved. Examplesof pH_(i),-raising agents are quaternary bases and their salts(carbonate, citrate, maleate), further TRIZMA (as base or as salt),THAM/tromethamine/trometanol, mono-, di- and triethanolamine, etc. Anintracellular pH-rise to 7.3-7.6 is the prerequisite for cellproliferation. The O₂-deficit is associated also with prostaglandininstead of leukotrien-synthesis. In order to down-regulate theimmunosuppressive effect of hyperactivated macrophages onimmunocompetent cells in inflammatory tissue (tumor, persistentinfection etc.), the following steps are recommended: (a) treatment byMabs and immunotoxins, directed against the surface antigens on enddifferentiated macrophages (x−4, x−11, x−12, x−14, x−15) and against thedifferentiation antigens on hyperactivated macrophages (b) treatment bycytotoxic substances, encapsulated in liposomes (c) treatment by agentswhich stabilize the lysosome-membrane in macrophages (e.g. goldpreparations such as aurothioglucose, aurothiopolypeptide orNa-aurothiomalate, further antimalaria-agents such as chloroquine andD-penicillamine).

[0185] The proteases are able to switch the cell from the suppressor- tothe effector function. The GEF (glycosylation enhancing factor), akallikreinlike kinin-protease prevents the GIF (glycosylation inhibitionfactor)-induced immune suppression; a membrane-associatedserine-protease is directly involved in the process of cell activation.

[0186] The lipases are able to activate -via lysophosphatideimmunocytes(macrophages, NK-, K-, T-cells). This activation results partly from themimicking of membrane-associated phospholipases (PLC, PLA2). The effectof mucopolysaccharidase is similar to that of proteases. Therefore, thedeblockade of immunocytes by proteases, lipases andmucopolysaccharidases is recommended, both per se or in combination withthe above described cell activators.

[0187] Interferons, TNFalpha and TGFB switch the cell from proliferationto dedifferentiation. Their pathologically increased levels exert anantiproliferative effect on precursor cells, depressing in this way therecruitment of immunocompetent cells. Such pathologically raised levelsof cytokines can be measured in AIDS- and tumor-patients, but also inautoimmune disorders and persistent infections. Therefore, theseantiproliferative cytokines should be neutralized by specific Mabs. Intumor- and AIDS-patients, the immune response can be improved by specialconjugates, consisting of the Fab/F(ab′)₂-subunit of tumor- orHIV-specific Mabs of murine origin plus the Fc epsilon-subunit of humanorigin.

[0188] Some membrane-associated immunorelevant structures (e.g. MHC II,MHC I, CD4, CD8, B2m) become immunosuppressive if they are shedded intothe plasma. The neutralization of such solubilized membrane structuresby specific Mabs helps to prevent their immunosuppressive effect.

[0189] A further advantage is the use of anti-HLA-DQ- andanti-LFA-lalpha Mabs.

[0190] The following procedure is of special interest:

[0191] (a) Tumor patients are first in vivo depleted of thetumor-protecting suppressor T cells (see patent applications DE3812605A1and PCT 94/EP89/00403.

[0192] (b) Patient's tumor cells are treated in vitro by IFN gammaand/or TNF allpha (postexpression of MHC II and MHC I), inactivated byanti-HLA-DQ- and/or anti -LFA-lalpha or their Fab/F(ab′)-subunit andreinjected into the patient. Alternatively, the tumor cells can befusioned (in the presence of PEG) with autologous and/or allogeneic MHCII-positive cells (B cells or macrophages)., treated by anti-HLA-DQ-and/or anti-LFA-lalpha-Mab and reinjected into the patient. Bothalternative procedures include the immune reactivation of the patient asan important step.

[0193] The principle of the here discussed novel tumor therapy is tocombine (a) the temporary breaking of resistance of immunocompetentcells with (b) the reinfusion of in vitro pregenerated tumor-specific Tcells. It is essential that these cells are memory cells; according tolaws of the so-called “restricted” CML (CML to non-MHC-molecules), onlythe blastogenically pretransformed T cells are able to induce in vitrothe response to the (soluble) antigen in question.

[0194] The tumor-specific T cells are capable to induce in vivo a kindof autoimmune reaction against the tumor antigen if they are generatedas follows:

[0195] (a) Patient's lymphocytes (T cells) are postexpanded in vitropolyclonally, e.g. by PHA, then the CD8-positive Ts cells are eliminatede.g. by specific Mabs or immunotoxins, and the residual CD4-positive Tcells are reinjected into the patient, along with the memory cells. Invivo, these T4 cells are able to induce the CTLs with specificity fortumor cells. Instead of the CD8⁺-postdepletion, the inducer-suppressorsubpopulation can be inactivated in advance by a 500 Rad-preirradiation.

[0196] (b) Alternatively, patient's lymphocytes (T cells) can beco-incubated in vitro with his malignant cells, after these have beeninduced to post-express the MHC II-antigen.

[0197] (c) An interesting further way comprises the reinfusion ofpatient's lymphocytes (T cells), along with mitomycin C-pretreatedpatient's macrophages.

[0198] The success of organ- and bone marrow transplantation can beessentially improved if the donor organ, more precisely its “passengerlymphocytes”. or donor bone marrow cells, respectively, are pretreatedin vitro by anti-HLA-DR- and/or anti-LFA-lalpha-Mabs (or theirFab/F(ab′)₂-subunit)

[0199] Alternatively or additionally, the MHC-postexpression on“passenger lymphocytes” can be depressed by Ca-antagonists (e.g.verapamil, nifedipin, diltiazem) or by anti-mitogenic substances(colchicin, domecolcin, gliotoxin). This MHC II- postexpression,following the organ surgery seems to be critical for the later organrejection. As mentioned above, the bone marrow cryopreservation can beessentially improved by the addition of PEG and/or PVP of dofferent molweight to DMSO.

[0200] An interesting novel approach in tumor therapy is the use ofhybridoma cells with restricted lifespan, constructed of tumor-specificimmunocytes and mitomycinC-pretreated immortalized partner cells. Onevariant proposes (a) to fuse the pregenerated immunocytes with myelomacells which are routinely used for hybridization purpose, and to treatthe resulting hybridoma cells with mitomycin C. (b) The otherpossibility is to fuse the pregenerated immunocytes withmitomycinc-pretreated myeloma partner cells, or(c) to use thecommercially available non-transformed fibro-blasts or embryonal cellsinstead of myeloma cells as partner cells. Instead of mitomycin C, othercell proliferation-limiting agents or a controlled cell irradiation canbe foreseen.

[0201] A typical mitomycin-concentration is 10 ug/ml, a typicalincubation time 18 hours (for 5 ug/106 cells). In this way, hybridomacells, based on tumor-specific plasma cells, CTLs, NK-, K/ADCC-cells andpreactivated macrophages can be constructed. They can be kept in cultureor frozen (PEG and/or PVP addition to DMSO); the preservation of thepreactivated state by this improved cryoprotective mixture is of specialrelevance for repeated infusion. The use of the HAT- or HAs-mediumguarantees a sufficient selection of the hybridoma cells; atime-consuming cloning is not necessary.

[0202] As next, some novel approaches in the treatment of cancer,chronic infections and autoimmune disorders, respectively, should bediscussed briefly.

[0203] (1) In tumor patients and patients with(chronic) infections,especially those with (retro)viral infections, the treatment withanti-B-cell-Mabs is recommended, since the level of immunosuppressiveICs is reduced and CTL-hindering, antigen-masking specific antibodiesare prevented to be synthesized. Examples of such Mabs are anti-CD19-,anti-CD20-, anti-CD21- and anti-CD22-Mab, as well as polyclonalanti-B-cell antibodies.

[0204] (2) Due to the negative impact of persistently activatedmacrophages on the progress of the disease, their elimination bymonoclonal and polyclonal antibodies, both in tumor patients and inpatients.. with chronic infections, including (retro)viral (HIV)ones,-is recommended.

[0205] Examples: anti-CD 15-, anti-CD 14-, anti-CD11c- and anti CD11b-Mates.

[0206] (3) The Mabs, described under points (1) and (2) can be injectedas coctail too.

[0207] (4) The points (1), (2) and (3) are valid also for the patientswith autoimmune disorders.

[0208] (5) The Mabs can be replaced by their conjugates with cytokines,radionuclids and/or cytotoxic agents to increase their efficacy.

[0209] (6) The Mabs can be also replaced by Fab/Fab′)₂-subunits,masking—not eliminating—B cells and macrophages.

[0210] (7) An essential progress in tumor therapy, according to theinvention, is the introduction of tumor cells with post-expressed MHCII, induced by interferons and/or TNFaipha. Before the reinjection,tumor cells are inactivated by mitomycin C, by heat or formaldebydeand/or glutaraldehyde.

[0211] (8) Alternatively, to point (7), hybridoma cells, arising fromthe fusion (in 20-40% PEG) of tumor cells with patient's MHC II-positivecells (macrophages, B cells), can be used. This cell fusion can becarried out with non-viable cells and doesn't include cell cloning.

[0212] (9) A further possibility is the presentation of tumor antigenplus autologous MHC II-antigen on the surface of liposomes.

[0213] (10) A new principle in tumor therapy is the mimicking of thestrong immune reaction, observed (a) during graft rejection and (b)during the autoimmune reaction. In both cases, the immune systemencounters the antigen (the allogen in organ grafts and the autoantigenin autoimmune reaction), first in its “processed” form, i.e. in contextwith the MHC II-complex. This favours the generation of Th and Tc cellsand depresses the Ts cells being able to interact with soluble antigensdirectly, i.e. without antigen processing. Therefore, the in vivosituation in graft recipients and patients with autoimmune disordersshould be mimicked in tumor-excised cancer patients by the injection ofpatient's tumor cells with the post-expressed MHC II-antigen. Anadditional improvement of the therapy is the use of the above-mentionedconstructs, consisting of the Fab/F(ab′)₂-subunit of tumor-specific IgGor IgM, plus Fc-subunit of IgE, alone or combined with IL4.

[0214] (11) As next, the in vitro preprocessing of patient'stumor-antigen is recommended; this occurs by the phagocytosis of thesoluble (e.g. 3M-KCI-extract) of tumor antigen or inactivated tumorcells, followed by the reinfusion of the involved macrophages into thepatient.

[0215] (12) The procedure, based on the generation of Th-memory cells bythe in vitro incubation of patient's T cells with his inactivated tumorcells, followed by the elimination of Ts cells through anti-CD8-Mabsplus complement, is of special interest. Both, the so pretreatedmacrophages and Th-cells mimic the situation during organtransplantation and autoimmune disorders which are characterized by astrong in vivo immune response. In this case, the in vivo immunereaction is directed against tumor cells.

[0216] (13) By the titration, i.e. “neutralization” of the solublefraction of immunorelevant membrane structures in plasma by specificMabs, the immunosuppressive effect of these humoral factors can beprevented. By the quantitative analysis, the patient-dependent amount ofthe neutralizing Mabs has to be determined in advance. Both the specificdiagnostic tests and the in vivo use of the neutralizing Mab aresubjects of this patent application. Examples of such molecules are theimmune-response-mediating or potentiating membrane receptors in general,e.g. IL2/Tac/CD25- and T11/CD2/Leu5a/IL4-receptor, further variousreceptor: ligand-systems of cooperating immunocytes (integrins such asCD2: LFA3, ICAM-1: LFA1), or immunorelevant structures in general, suchas CD4, CD8 and MHC II.

[0217] (14) The procedures, described under points (11) and (12) arealso valid for HIV-infected persons. Classical vaccines can beessentially improved as follows: (1) by the preformation ofantigen(pathogen): IgM-immunocomplexes which activates the macrophagesbefore Ts cells are stimulated (2) by the use of preformed complexes,consisting of pathogen: IgM/IgG: complement (or C-subunit) (3) by theuse of pathogen-specific IgE and/or conjugates of pathogen-specificFab/F(ab′)₂ subunit of IgG/IgM plus Fc-fragment of IgE (of anyspecificity) (4) by combining the priming with anti-CD8-Mab plusanti-B-cell-Mab and the boosting with anti-CD8-Mab alone (withoutanti-B-cell-Mab) (5) by masking the macrophages/monocytes with theFab/F(ab′)₂-subunit of the specific Mabs, after the first or during thesecond vaccination (6) by injecting Mabs, neutralizing gamma-interferon,TNFalpha, PGE2 and/or TGFbeta (−+IL4) 4-6-days after the first or duringthe second vaccination.

[0218] The next point is the prevention of tumor relapses after BMT bythe potentiation of the GvL-effect, without the danger of the parallellyincreased GvH-reaction. After years of research, several procedures,based on the circumvention of the critical, often fatalimmunosuppressive. phase immediately after the tumor removal, have beenworked out. These procedures can be used per se or in combination withother techniques, described in this patent application.

[0219] One procedure comprises the admixing of donor lymphocytes, whoselifespan has been predetermined (preprogrammed) by a special3-step-procedure, to the donor bone marrow cells. In this way, thepatient gets immunocompetence, until he restores his own immuneresponse; he is continuously able to combat infections and to preventthe reinduction of tolerance against residual tumor cells and herewiththe later relapses. Before the donor T cells can provoke theGvH-disease, they die due to the preprogrammed cell death. The celldeath can be predetermined by a controlled in vitro pretreatment ofdonor T cells with DNA-crosslinking agents. The preprogrammed cell deathof immunocompetent donor T cells can be achieved also—via theintracellular irradiation—by the in vitro incorporation of radiolabeled,i.e. radionuclid/radioisotope-containing nucleosides, nucleotides, freebases and their derivatives into the DNA. Examples of such radiolabelednucleosides are: 2′-deoxyuridine-2-′¹⁴C, further 2′-deoxyuridine-5-³,2′-deoxycytidine-S-³H and 5-bromo-2′-deoxyuridine-2-′¹⁴C. Allnucleosides and nucleotides, labeled by various redionuclides (e.g. fromH, P, S and other) and added to the culture medium, are subject of thispatent application.

[0220] If donoe's immunocompetent T cells are coincubated with therecipient's mitimycinc- or irradiation-inactivated MHC II (and/or MHC1)-positive cells, the cell death-preprogramming radiolabelednucleosides are incorporated nearly selectively into the alloreactive,recipient-recognizing donor T cells.

[0221] In the special case of the radiolabeled bromo-deoxyuridine, theprelabeled immunocompetent donor's T cells, added to the T-depleteddonor-bone marrow, can be treated after the restoration of patient's ownimmune system ex vivo by UV-irradiation to support additionally theself-destruction of these cells by radio labeling. A similar techniqueof radionuclide-incorporation into the cell nucleus can be used,according to the invention, for killing of tumor cells in situ. Here,the carriers of the radioactive, tumor-destroying radiation are nottumor-specific Mabs but (a) the tumor-recognizing TIL (tumorinfiltrating lymphocytes), and (b) the tumor-specific Tc/CTL(cytotoxic Tcells).

[0222] A special advantage of the cellular over the molecular, i.e.Mab-mediated transport of the tumor-destroying radionuclides into theimmediate neighborhood of tumor cells is the essentially intensity(density) of the cellular irradiation source. The selectivity (a) of TILcells as carriers of radioactivity is guaranteed by thetumor-specificity of TIL-cells (Rosenberg, Anderson, Blaese), and (b))that of tumor-specific Tc/CTL by their selective recognition of tumorantigens (TATA, TSTA, TSA). Though TIL cells belong both to the CD4-andto the CD8-positive T cells, a clear cut-off between TIL- andTc/CTL-cells is not possible.

[0223] The preparation of TIL- and Tc/CTL-cells as carriers oftumoricidal irradiation includes radiolabeled amino acids and otherradiolabeled cellular components (precursors), in addition to theradiolabeled nucleosides; they must be added to the culture medium andare incorporated into TIL- and/or Tc/CTL-cells.

[0224] A further possibility of bridging the fatal immunosuppressivephase after the removal of primary tumor or after following the BMT isthe in vitro pretreatment of donor's immunocompetent cells byphotosensitive dye-stuffs (e.g. psoralen), followed by their addition tothe donor's T-depleted bone marrow, and their transfusion into therecipient. Later, after having protected the patient (recipient) frominfections and from the clonal expansion of residual tumor cells, thesephotolabeled cells are selectively eliminated ex vivo by UV-irradiation.The used technique (photopheresis/PUVA) profits of predeveloped devices.For efficiency reasons, the combination of this technique with otherhere described techniques is recommended.

[0225] In analogous technique, photosensitive dye-stuffs are combinedwith or replaced by the radiolabeled or non-labeled bromo-deoxyuridine;again, the labeled cells are selectively killed ex vivo by theUV-irradiation. The use of radioactive instead of the cold isotope inBrdUr increases the efficacy of the technique. It should be combinedwith a preincubation of donor immunocompetent cells with inactivated MHCII (and MHC-1)-positive recipient cells which allows a selectivepreprogramming of cell death in the recipient-specific alloreactivesubpopulation of the donor.

[0226] A further way is the in vitro transfection of donor'simmunocompetent T cells with strongly immunogenic surface antigens,followed by their addition to the T cell-depleted donor bone marrow andby later transfusion into the recipient (e.g. tumor patient). The laterin vivo elimination of the mature donor T cells which helps to bridgethe immunoincompetent phase is carried out by antibodies, directedagainst the transfected membrane antigen. These antibodies can beconjugated with cytotoxins in order to increase their efficacy. Afurther modification of this technique is based on the addition of Tcells of a second MHC-incompatible donor to the T depleted bone marrowof the first donor. After the restoration of patient's ownimmunocompetence, mature histoincompatible donor T cells are eliminatedin vivo by allotypic antibodies or corresponding immunotoxins. All thesetechniques can be used alone or in different combinations. The nextprocedure is based on the cumulative (additive) effect of DNA-damagingby cytotoxic agents and/or irradiation. The immunocompetent cells of thedonor are treated first in vitro by the sub-lethal dose of the DNAdamaging cytotoxic agent and/or irradiation and later in vivo after thefinished mission (bridging of the fatal immunosuppressive interphase,before the restoration of patient's immune competence) by a second(lethal) dose of cytotoxic agent(s) or irradiation. The strongGvL-effect of the so pretreated donor bone marrow can be furtherintensified if the donor's immunocompetent T cells to be admixed to thedonor bone marrow are predepleted of alloreactive subclones andactivated in a tumor-specific or non-specific way, before they have beeninjected into the patient. In addition, the technique, based on a noveltype of immunocompetent T cells which is characterized by a “frozen”activated functional state, should be described briefly. Details aboutthis novel cell type, implicating a constitutively activated statewithout cell proliferation, follow below. Cells of this novel type canbe added to T-depleted donor bone marrow and must not be later oneliminated in vivo, because their ability to proliferate is geneticallyswitched off. Characteristically for all these techniques is (a) astrongly increased GvL-effect, without a simultaneous GvH-reaction, (b)opening of new ways for histoincompatible BMT, (c) the option of anadditional tumor-specific and/or non-specific preactivation ofimmunocompetent T cells, admixed to the T-depleted donor bone marrow,and (d) the impact on both the GvH- and HvG-reaction which minimizes theBMT-associated complications.

[0227] These facts support the inventor's idea to introduce BMTobligately in the patients with solid tumors. According to theinvention, the classical BMT could be replaced by the simple exchange ofpatient's (recipient's) T cells by T lymphocytes of the donor inpatients with solid tumors. This can be carried out by an in vivodepletion of patient's T cells by specific Mabs or immunotoxins andtransfusion of healthy donor T cells; the histoincompatibility problemscan be overcome by the above described special pretreatment ofimmunocompetent T cells.

[0228] According to a further therapeutic model, the bridging of thefatal immunosuppressive phase, following the tumor excision or BMT canbe achieved by autologous or allogeneic LAK-cells plus rIL-2. SinceLAK-cells consist of ca. 90% NK-cells and of ca. 10% non-MHC-restrictedCD3⁺-T cells, the in vitro predepletion of CD3⁺, cells is recommended inthe case of allogeneic LAK-cells. Alternatively, the completeLAK-population can be treated by one of the above described celldeath-preprogramming procedures. The so pretreated allogeneic LAK cellsare added to the donor bone marrow before their injection into therecipient. An important positive “side effect” is the in vivo killing ofrecipient's HvG-inducing residual T cells. In this way, thecomplications of a radical recipient conditioning could be prevented.This HvGR-inhibiting effect is based on the property of NK- andLAK-cells to recognize and to inactivate—via the4F2/TNKTar-antigen—fastly proliferating cells. The plasmapheresis bringssome additional advantages in the autologous or allogeneic BMT. Theremoval of immunosuppressive factors, belonging primarily to the immunecomplexes, to the solubilized cytokine- and growth factor-receptors, aswell as to the prostaglandins, has been neglected in conventional BMT. Afurther improvement is the addition of fibroblast to the donor BM. Thesituation following BMT shows a common element with the situation ofimmunocytes in the limiting dilution-test, in which the addedfibroblasts, by secretion of growth factors, make the growth and thesurvival of immunocompetent cells possible.

[0229] Though there are trends to shorten the phase ofimmunoincompetence in tumor patients and BM-recipients by cytokines(G-CSF, GM-CSF), fibroblasts, admixed to the donor bone marrow areexpected to secrete a much broader spectrum of cytokines.

[0230] As next, the addition of donor-macrophages to donor-BM isrecommended; in this way, the cooperation of accessory cells (APC) withT cells in the critical post-transplantation phase is guaranteed. Thetemporary masking of recipient-macrophages (to inhibit the preexistentsuppressor monocytes) is also of interest.

[0231] The next point is the selective in vivo depletion of therecipient macrophages/monocytes (e.g. by specific Mabs or immunotoxins)and/or the addition of donor-macrophages to donor bone marrow. Finally,the use of neutralizing Mabs, directed against all those cytokines whichallow the mutual activation of macrophages, T4 cells and NK cells in thecritical phase of the GvH- and HvG-reaction, is recommended. So, theclonal expansion of alloreactive subpopulations, following allogeneicbone marrow and organ transplantation, can be prevented byneutralization of TNF alpha, IL-1, IL-6 and/or gamma IFN. As next, anovel cell type should be described which is characterized by the“frozen”, i.e. constitutively activated functional state and by aparallel switching off of the cell proliferation. This state isassociated with the cell arrest in the G₁- or G₂-phase and with apermanently increased level of the intracellular Ca²⁺(Ca₁). In the caseof ex vivo generated LAK- and TlL-cells, the problem of a rapid activitydrop of these cells in vivo could be solved by the corresponding cellmodification. This in vivo inactivation of LAK- and TlL-cells stems fromthe induction of lipocortin/lipomodulin by plasma corticosteriods(cortisol) and can be prevented in two ways: (1) by the constitutive,proliferation-free activation of LAK- and TlL-cells, and (2) by thetransfection of LAK- and TIL-cells with the CDNA, encoding thecortisol-cleaving enzymes, such as 20alpha-hydroxysteroid-dehydrogenaseand B-glucuronidase.

[0232] The T cell-activation (a) by different cytokines, such as IL-2,IL-3 and CSF-1/M-CSF, and (b) by (processed) antigen occurs in the sameway, i.e. via the activation of the PI (phosphoinositol)-dependent PLC(phospholipasec) and results in the increase of the intracellularCa,²⁺-level.

[0233] The constitutively increased Ca,²⁺-level in the cellcycle-arrested cells (subject of this patent application) opens a novelway of cell activation. A special advantage is the cell activation andthe maintaining of this activated state even in the absence of thespecific signal, e.g. the processed antigen in the case of helper Tcells. In other terms, the constitutively ^(increased cai2+)-level inthe cell cycle-arrested cells confers to these cells the geneticallypredetermined (highly specialized) function, e.g. the production ofspecific antibodies or the CTL- or ADCC-activity. A further advantage isthe inner stability, i.e. the resistance against the specific ornon-specific suppression by suppressor factors, such as prostaglandins(e.g. PGE1/E2) or corticosteroids, without the danger of an uncontrolledcell proliferation (because of the cell cycle-arrest).

[0234] This goal can be reached in two ways: (a) by hybridization ofcells with the desired function, e.g. plasma cells, CTL/Tc, T4/Th etc.with immortalized cells (autologous tumor cells of the patient ortransformed cells of other origin), followed by the treatment of formedhybridoma cells by cell death-preprogramming techniques, as describedabove, and (b) by a double transfection of the cells, showing thedesired function (B, T4, T8), with the sense-cDNA, encoding theconstitutive cell activation plus antisense-cDNA, encoding the switchingoff of cell proliferation.

[0235] Ad (a): The cell death-preprogramming treatment can occur eitherin hybridoma cells or in immortalized partner cells, before these havefused to give hybridoma cells.

[0236] The rejection of hybridoma cells, based on non-allogeneicimmortalized cells, must be prevented (a) by their repeated incubationin the presence of the alloantigen-specific antibody, or (b) bytransfection of the cells with the histospecific antisense-cDNA.

[0237] Candidates for the immortalized partner cells are thosetransformed cells which maintain an increased intracellular Ca_(i)²⁺-level by autocrine or paracrine mechanisms or such transformed cellswhich are able to maintain—independently of external signals—theactivated state either by the constitutive, ligand-dependent activity oftyrosine- or serine/threonine-kinases or by the continuing PI-conversionto PIP2.

[0238] Ad (b): The double transfection of the target cells, showing thedesired function (B,T4,T8 . . . ) (b1) with sense-cDNA, encoding theconstitutive expression of cell-activating signals, plus (b2) withantisense-cDNA, encoding the turning off of cell proliferation, createsa permanently “turned on”, non-proliferating cell which doesn't need anyspecific signals (e.g. processed antigen) for its activation.

[0239] These novel cell constructs have an enormous practical relevance.Examples are (1) the intracranial injection of NGF(nerve-growth-factor)- and/or DOPA-secreting, long-lasting,fibroblast-based cells constructs which cannot be down-regulated byplasma suppressor factors and are of special interest in the treatmentof M. Alzheimer and M. Parkinson, further (2) the cytokines and growthfactors-producing cell constructs which support patient's immune system,following high-dose (radio)chemotherapy and BMT, as well as (3) cellconstructs, based on tumor-specific plasma cells which are able toproduce anti-tumor-Mabs in situ, preventing in this way the induction ofneutralizing immunoglobulins.

[0240] As the sense-cDNA, encoding cell activating signals (1) the cDNA,encoding various cytokines or (hematologic) growth factors and/or theirreceptors, further (2) the PLC- and PLA2-encoding cDNA, (3) the cDNA,encoding different cytoplasmic serine/threonine-kinases, and (4) thecDNA, encoding various protein-kinases, such as C-kinase,Ca/calmodulin-kinase, casein-kinase II and G-kinase, are recommended.

[0241] The prevention of cell proliferation can be achieved by theantisense-cDNA of all cell division-inducing factors. Examples are (a)cyclinA, cyclinB 1, c-ras, c-raf, PSTAIRE, MPF, p34^(cdc2), pl3, further(b) the DNA-transcription factors like AP-1 (AP-I) and AP-2(AP-II), and(c) DNA-polymerase-alpha, PCNA and (protein)elongation factor(elF-2/elF-2p). PSTAIRE is a cdc2-subregion (aminoacids 42-56) andbelongs, like p34^(cdc2), to the family of cell cycle-specificprotein-kineses. The PCNA (proliferating cell nuclear antigen)is a36kD-intra-nuclear-polypeptide and component of polymerase-delta; theMPF stands for mitose- or M-phase-promoting-factor.

[0242] Alternatively to these antisense-cDNAs, various sense-cDNAs,encoding suppressor-oncogenes, such as plO5RB and p53 can be transfectedto prevent uncontrolled cell proliferation. By transfection ofextra-copies of the p53 and/or RB-cDNAs,somatic cells, includingleukocytes, can be made more resistant against different carcerogenicagents.

[0243] The uncontrolled proliferation can be switched off also by cellfusion with normal cells, expressing the wild type or the wild-type p53.

[0244] One of the points, according to the invention, is the preventedexpression of the MHC II or MHC I complex on the cell construct,achieved by the transfection of the MHC II or MHC I-encodingantisense-cDNA. The antisense-cDNA can be replaced (a) by ribozymes, (b)by psoralent derivatives of the antisense-oligonucleosidesor—oligonucleosidemethylphosphonates, and (c) by antigen—andantisense-oligonucleotid-intercalator-conjugates.

[0245] (a) The advantage of ribozymes, called also “catalytic RNA”, overthe corresponding antisense-cDNA is their irreversibility as they cleavethe sense-DNA. The smallest and simplest self-cleaving domain ofribosome is the “hammerhead”-structure, e.g. the “structure I”,described by Uhlenbeck or the “form IV”, reported by Haselhoff andGerlach.

[0246] (b) The psoralene-derivatives ofantisenseoligonucleosidmethylphosphonates are also irreversible in theiraction; here, the desired sense-DNA is switched off by the photoinducedDNA-crosslinking.

[0247] (c) When the technique, based onoligonucleotid-intercalator-conjugates is used, the irreversibility isachieved by the conjugation of antisense-cDNA or -RNA with thechemically (Cu²⁺-phenanthroline) or photochemically (ellipticin)inducible intercalator-molecules.

[0248] The immunotherapy of malignancies and autoimmune disorders, aswell as of bacterial and (retro)viral infections is based on the sameprinciples as the improvement of conventional vaccines and prevention ofgraft rejection, namely (a) on the deblockade of the hyperactivatedstate of immunocompetent cells and/or (b) on the elimination orinactivation of hyperactivated effector cells (“microimmunosurgery”).(a) This deblockade of the hyperactivated state of immunocytes can beachieved, according to the invention, by combining agents which blockthe “voltage-operated” and/or “receptor-operated” Ca²⁺-channels(“component I”), with agents which reduce the intracellular cAMP or thecAMP/cGMP-ratio, respectively (“component II”).

[0249] The “component I” comprises “classical” Ca-antagonists(Ca-channel-blockers) of all subtypes, e.g. phenylalkylamines,dihydropyridines, benzothiazepines, piperazines, quinoxalines,quinazolines (e.g. bepridil and perhexilin). Alternatively oradditionally, a parallel inhibition (blockade) of alpha-plus Badrenoceptors, of H2-plus H1-histamine receptors, of A2-plusAl-adenosine receptors, of 5-HT/serotonine) receptors and/or receptorsof various inflammation mediators (e.g. bradykinin, kinin-cascade,complement-cascade, especially c5a, c4a, c3a, PAF etc.) is recommended.Examples of preferred combinations are listed below; they can becombined with sub-dosed nitro-compounds, including molsidomine,Ca-overload.blockers, e.g. cinnarizine, Ca-antagonists, BRM and/orcytokines. The combination of molsidomine and nicerogoline (analpha-blocker), with or without Ca-overload-blocker(s) (e.g.cinnarizine)is of a special interest.

[0250] The “component II” comprises cAMP/cGMP- or cAMP-reducing agents.Examples are antagonists of all cell receptors which are coupled via theGs-protein to the membrane-associat,ed adenylate cyclase (AC). Thiseffect is increased if agonists of G_(p)-, G_(i)- orG_(o)-protein-coupled receptors and/or of cGMP-increasing, Ca_(i),reducing nitro-compounds (e.g. isosorbid-mono- and -dinitrate,glycerol-trinitrate/nitroglycerol, erythrit-tetranitrate,pentaerathrit-tetranitrate, amylnitrite, molsidomine) are givenparallelly. The antagonists of those Gs-coupled receptors whose normal,physiological agonists bind at the same time P_(p)-,G_(i)- or G_(o)-coupled receptors are of a special interest. After having blocked the.Gs-coupled receptors, the Gs-coupled receptors, the endogenous ligandbind the G_(p)-, G_(i)- or G_(o)-coupled receptors to a higher degree.In this way, B-blocker achieve 2 effects, the cAMP-drop and thecGMP-rise in cytosol of immunocompetent target cells. Examples of suchendogenous agonists are catecholamines (adrenaline and nonadrenaline),histamine and adenosine. So, the antagonists of B-adrenoceptor (onimmunocytes, e.g. T cells and monocytes/macrophages) causecatecholamines, primarily adrenaline, to interact with the alpha-instead of B-adrenoceptor. Similarly, the antagonists of H2-histaminereceptor make the histamine ligate the cAMP-lowering H1- instead of thecAMP-increasing H2-receptor. In the presence of A2-antagonists, theendogenous agonist adenosine binds to the cAMP-depressing A1- instead ofto the cAMP-raising A2-subtype P1-adenosine receptor.

[0251] It is also advantageous to use the agents which inhibitreversibly both, the Ca- and the Na-channels. The working mechanism isthe increase of the resting potential of immunocompetent cells which isdecreased during the hyperactivated state. This class of substancescomprises (a) some Ca-antagonists like cinnarizine, flunarizine,fendiline, bepridil, tiapamil and partly verapamil and gallopamil,further (b)sub-dosed antiarrhytmics (class I to IV), especially thecombination of class IB with class III, due to the equilibrated K-efflux(class IB) and K-influx (class III) and synergism in the inhibition ofNa-channel (among these agents are adrenoceptor-blockers sotalol andpropranolol). The combination of cinnarizine and propranolol is of aspecial interest.

[0252] The central point of this invention is the combination of agentswhich decrease the cAMP-level or the cAMP/cGMP-ratio with those whichblock the “voltage-operated” and/or “receptor-operated” Ca-channels. Inspecial cases, the “component I” and “component II” are identic. So,some special nitro-compounds, such as syndnonimine derivatives (e.g.mosidomine), show both effects (cGMP-rise, Ca_(i)-decrease); they arealso subject of this invention.

[0253] Both, the therapy of the diseases discussed above and theefficiency of conventional vaccines can be further improved bymethylxanthines, by pHi-increasing substances, ba redox-potential,GSH/GSSG and NADP)H/NAD(P)+ correcting agents, as well as by ionichomeostasis and K-balance influencing substances.

[0254] (b) Inactivation/elimination of hyper- or persistently activatedimmunocompetent cells.

[0255] The principle of this in vivo inactivation or depletion ofhyperactivated effector cells is the “microimmunosurgery”, a newtechnique which would compete with or complement the gene-therapy infuture. The principle is the selective inactivation of disease-inducingand disease-maintaining lymphocyte-subclones by the combination of (b1)panT- or T subclass-specific Mabs or Mab-derived immunotoxins, plus (b2)alloreactive-T cells of a healthy donor. This combination of humoral(Mab) and cellular (allogeneic T cells) technique is able to increaseessentially the efficiency of the Mab-mediated depletion of pathogenicimmunocyte-subclones, as shown in animal model. Tumor patients as wellas patients with (chronic) infections (including HIV) and those,suffering from CFS (chronic fatigue syndrome) show a strongly increasednumber of CD8-positive (Ts) and/or HLA-DR(MHC II)-positive T cells. Bythe combination of anti-CD3- or anti-CD8-Mabs plus allogeneicdonor-PBM/PBL or donor T cells whose cell death is“preprogrammed”/“predetermined” by a special in vitro-treatment, themeotioned pathologic (hyperactivated) CD8:HLA-DR-positive effector cellscan be selectively eliminated in vivo. In this way, a 94-100%-survivalrate in tumor-bearing mice could be achieved.

[0256] The “preprogramming” of cell death in allogeneic donor PBM/PBL orT cells is a multistep in vitro procedure, comprising the followingsteps:

[0257] (1) Synchronization of “donor effector cells” (a) by theirincubation in serum-free medium, followed by the incubation inserum-containing medium, or (b) by cell incubation first in the absenceand later in the presence of essential aminoacids (e.g. isoleucin), or(c) by cell incubation in the presence of synchronizing cytotoxicagents, such as vinchristine, hydroxyurea or bleomycin.

[0258] (2) Treatment of “donor-effector cells” by (a) bifunctionalalkylating agents, i.e. DNA-cross-linking agents (e.g. mitomycin C)and/or (b) by inhibitors of the enzyme ribonucletoid-reductase (e.g.hydroxyurera) which block the DNA- but not the RNA- orprotein-synthesis.

[0259] (3) Incubation of “donor effector cells” in the presence ofinhibitors of DNA-reparases (DNA repair system) (e.g. hydroxyurea).

[0260] (4) A thorough washing of cells, e.g. by PBS or RPMI 1640.

[0261] (5) Infusion of so pretreated “donor effector cells” into therecipient (e.g. tumor patient).

[0262] An alternative procedure consists of the incubation of “donoreffector cells” (a) with radiolabeled DNA-constituents (purine- andpyrimidine bases) and/or (b) with radiolabeled aminoacids.

[0263] A further alternative is the irradiation of “donor effectorcells”, followed by their incubation in the presence of inhibitors ofDNA-reparases (e.g.hydroxyurea).

[0264] In the special case of autoimmune disorders, the target cells of“preprogrammed” alloreactive donor T cells are also the HLA-DR(MHCII)-positive autoaggressive T cells (mostly T4, partly T8 cells).

[0265] The procedure consists of the in vivo depletion of T cells (byanti-panT/CD3-Mabs) or their T8 and/or T4 subclass. Because of the highcosts for the “pure” Mabs and Mab-derived immunotoxins, theircombination with cytotoxic agents, especially cyclophosphamide isrecommended.

[0266] A further improvement of the technique is the reinfusion ofpatient's peripheral blood cells which have been preactivated ex vivoagainst the donor PBMs/PBLs, into the patient, after the premanipulateddonor effector cells have eliminated the pathologically activatedrecipient lymphocytes.

[0267] As animal experiments have shown, the efficiency of“microimmunosurgery” is so high that the donor effector cells are ableto lyse patient's pathologic leukocyte-subclones even (a) without apreceding in vivo Ts-depletion (by Ts- and/or panT/CD3-specific Mabs),though the combined attack on the cellular and humoral level remains themost efficient approach. All experiments, however, show an enormous risein their efficacy when the in vivo used Mabs are combined with the celldeath-preprogrammed effector cells. This concerns the therapeuticallyused anti-tumor-Mabs, the Mabs, utilized in the recipients of organgrafts (e.g. anti-CD3-Mabs) and the Mabs, used in the treatment ofautoimmune disorders (e.g. anti-CD4-Mabs).

[0268] A further possibility (a) to bridge the criticalimmunoincompetent phase, e.g. after the excision of primary tumor orfollowing the BMT, and (b) to prevent GvHR (but not GVLR), is asfollows:

[0269] (a) Patients, e.g. those with solid tumors, are first“conditioned” by anti-CD3-Mab (or the corresponding immunotoxin), or bythe Mab-saving combination of cytotoxic agent (e.g.cyclophosphamide=plus Mab

[0270] b) In addition, the CD8:HLA-DR/DQ-double positive suppressorfraction is eliminated by means of “microimmunosurgery”.

[0271] (c) The next step is the in vivo depletion of donor effectorcells by patient's (i.e. autologous) T cells or PBMs/PBLs. The ratiobetween the autologous T cells or PEMs/PBLs (point (c)) on the one handand the corresponding leukocyte-subpopulations of the healthy donor(point (b)) on the other hand should be 3:1 to 10:1; in this case, thecell death-programming can be omitted. Both, the donor effector cells(point(b)) and the autologous effector cells (point (c)) can, but mustnot be prealloactivated. It is advantageous if parental PBMs/PBLs areused as donor effector cells.

[0272] The presensibilization of donor alloreactive effector cellsagainst the recipient lymphocytes brings an additional advantage: The sopresensibilized (primed) donor effector cells are blastogenicallypretransformed and can be activated in vivo, in some analogy to theeffector cells of the secondary MLC and those of the PLT 1-2 daysearlier as the non-primed clones. This fact confers to thepresensibilized (primed) donor cells the crucial advantage, that theyare able to eliminate—following their infusion into the patient—hispathologically (hyper)activated, MHC II-positive subpopulations beforethe patient's defense against these therapeutically utilizeddonor-effectors can be organized. For this reason, the number of donoreffectors to be transfused can be reduced and the celldeath-preprogramming possibly omitted.

[0273] (c) Improvement of organ grafts (kidney-, heart/lung-,liver-allografts)—The acute rejection, leading to the loss of the graftorgan can be essentially improved, according to the invention, if theclassical procedures, based (a) on a generalized immunosuppression(azathioprine, prednison, (methyl)prednisolon, cyclosporinA), and/or (b)on the in vivo T cell depletion by anti-CD3-Mabs or ALG or ATG, arecompleted or partly replaced by the “microimmunosurgery” or by the celldeath-preprogrammed effector cells.

[0274] (d) Improvement of conventional vaccines—The same principle(deblockade and/or inactivation /elimination of hyperactivated effectorcells) can be used to improve the conventional antibacterial andanti(retro)viral vaccines, including the anti-HIV-vaccines; the targetcells are here the CD8: MHC II-double positive suppressor effectorcells. The principle is the increase of the absolute number ofblastogenically pretransformed, pathogen-specific Th-, Tc/CTL- and/orB(plasma) cells by (a) deblockade and/or (b) inactivation /eliminationof pathogen-specific Ts cells. In an early phase, these Ts inhibit thegeneration of pathogen-specific Th-, Tc/CTL- and B-memory cells. By (a)deblocking and/or (b) inactivating/eliminating the Ts-cells, the clonalexpansion of “positive” memory cells (Th, Tc, B) is strongly increasedand the protection of infection significantly improved.

[0275] (a) The deblocking of Ts-cells occurs by the combination of“component I” and “component II”

[0276] (b) The inactivation/elimination of CD8:HLA-DR/DQ-positiveTs-cells is performed by alloreactive, cell-death-preprogrammed effectorcells (see above)

[0277] (c) The “immunologic memory”, induced in a pathogen-specific wayby vaccination, can be further improved by eliminating in vivo thepathogen-specific Ts-cells of the vaccinated person with subset(CD8/Ts)- and/or panT(CD3)-specific Mabs or corresponding immunotoxins

[0278] (d) The number of pathogen-specific Th-, Tc- and B-cells can beincreased also by delaying the antibody-production through Mabs (andimmunotoxins), directed against (d1) B-cells, (d2) Th(T4)-cells, (d3)B-plus Th(T4)-cells, (d4) monocytes/macrophages (“suppressormonocytes”), and/or (d5) B-cells plus suppressor-monocytes.

[0279] Again, “pure” Mabs can be replaced by the Mabs-saving mixture ofcytotoxic agents (e.g. cyclophosphamide) plus Mab.

[0280] (e) Support and partial replacement of glucocorticoids (e.g.cortison) by low-dosed antagonists of Gi(Gp,Go)-coupled receptors and/orby low-dosed agonists of Gs-coupled receptors

[0281] Glucocortocoids, characterized by strong side-effects, can besupported or partly replaced by sub-dosed blockers of Gi-, GporGo-coupled receptors and/or by low-dosed ligands of Gs-coupledreceptors. These agents can be combined with sub-dosed Ca-antagonists.

[0282] The indication of these novel combination preparationscorresponds to that of “classical” glucocorticoids. Again, theantagonists of those Gi(GP, Go)-coupled receptors which share theendogenous ligand with a Gs-coupled receptor (e.g. catecholamines,histamine, adenosine) are of a special interest. With such combinations,other clinically used immunosuppressive agents (e.g. cyclosporine,FK506, rapamycin, azathioprin, cyclophosphamide) can be supported orpartly replaced.

[0283] Since the hyperactivated state of effector cells (T cells,macrophages etc.) is an important element in the pathogenesis (a) ofcancer (b) of autoimmune disorders (c) atherosclerosis (d) infectiousdiseases (including HIV), the procedures, described under the point (a)(“Deblocking of hyper- or persistently activated immunocompetent cells”)and under the point (b) (“Inactivation/elimination of hyper- orpersistently activated immunocompetent cells”) are valid for all thesediseases.

[0284] The reduction of background-signals, i.e. “filtering out” ofnon-specific (non-productive) transmembranal signals increases thesusceptibility of immunocompetent cells for specific, immunorelevantsignals.

[0285] 1 Combination of sub-dosed B-(B1- plus b B2-) and sub-dosedalpha-(alphal- plus alpha2) adrenoceptor blockers (antagonists)(objective: prevention of hypoergic or anergic state of hyperactivatedimmunocompetent cells by lowering the level of non-specificbackground-signals).

[0286] 1.1 Combination of preparations on pindolol-basis (e.g.durapindol/−15/-retard (26.039) or pinbetol/forte (26.067) pluspreparations on phenoxybenzamine-basis (e.g. dibenzyran 1/5/10(81.091)).Pindolol is a B1- plus B2-sympatholytic, phenoxybenzamin an alphal- plusalpha2-blocker. Recommended dose: 1×15 mg/d or 3×5 mg/d or 2-3×1 mg/ddibenzyran.

[0287] 2 Combination of sub-dosed B-(B1- plus B2-)-adrenoceptor-blockerswith sub-dosed alphal-receptor-antagonists, blocking at the same timethe H1-histamine and the 5-HT(serotonin) receptor (objective: see point(1)).

[0288] 2.1 Combination of drugs on pindolol-basis (e.g.durapindol/−15/-retard (26.039), or pinbetol/forte (26.067) pluspreparations on indoramin-basis (e.g. wydora/50(16.039)). Indoramin isthe antagonist of alphal-adrenoceptor, of H1-histamine receptor and of5-HT receptor. Recommended dose: durapindol (see above); indoramin 1×25mg/d.

[0289] Combination of sub-dosed B- (B1- plus B2-)adrenoceptor-antagonists with sub-dosed alpha- (alphal- or alpha2-)receptor blockers (objective: see point (1) and (2)).

[0290] 3 Combination pf preparations, based on 3.1.1. alprenolol (e.g.aptin (26.002)), 3.1.2. bupranolol (e.g. betadrenol 50/−100(26.017)),3.1.3. penbutolol-sulfate (e.g. betapressin (26.019)), 3.1.4.bisoprololfumarat (e.g. concor 5/10 (26.025)) or 3.1.5. carteolol (e.g.endak 5/10 (26.046)) plus preparations, based on 3.1.1. urapidil (e.g.ebrantil 30/60/90 (16.032)), 3.1.2.(doxazosinmesilate (e.g. cardular 1mg/-2 mg/-4 mg (16.029) or diblocin 1mg/-2mg/-4mg (16.030)) or 3.1.3.terazosin (e.g. heitrin 1/2/5 (16.035)). Recommended doses:aptin-duriles 1×200 mg/d; betadrenol 1-2×50 mg/d; betapresin 0,5-1×40mg/d; concor 1×5 mg or 1×10 mg/d; endok 5 mg/d; ebrantil 30 mg/d,cardular 1 mg/d; diblocin 1 mg/d; heitrin 0,5-1 mg/d.

[0291] 4 Combination of sub-dosed H2- and Hi-histamine receptorantagonists (objective: see above).

[0292] 4.1 Combination of preparations, based on cymetidine (e.g.sigacimet 200/−400/−800 (59.102) or tagamet 200/−400/−800 (59.105) orH2-blocker ratiopharm (59.096) plus preparations, based on 4.1.1.oxatomid (e.g. barpet (07004)), 4.1.2. bromopheniramine-hydrogenmaleate(e.g. dimegan (07.005)), 4.1.3. dimetindenmaleate (e.g. fenistil(07.006)) or 4.1.4.terfenadine (e.g. hisfedin (07.009)). Recommendeddoses: sigacimet 1×200 mg/d; tagamet 1×200 mg/d; H2-blocker ratiopharm1×200 mg/d; barpet 1×30 mg/d; dimegan 1×12 mg/d; fenistil 1×1 mg/d;hisfedin 0.5-1×60 mg/d.

[0293] 5 Combination of sub-dosed antagonists of A2- plusA1-P1-purinergic (adenosine) receptors (objective: see above.

[0294] 5.1 Combination of preparations, based on methylxanthines(theophylline) (e.g. aerobin mite (27.102) or contiphyllin retard(27.113) or euphyllin N (27.125)) plus preparations, based onipratropium-bromide (e.g. atrovent (27.048) or itrop (09.028)).Recommended doses: aerobin 0.5-1×200 mg/d; contiphyllin 0.5-1×300 mg/d;euphyllin N 1×73mg/d; atrovent 0.5-1×200mg/d; itrop 0.5-1×10 mg/d.Combinations, based on cromoglicinic acid (diNa-salt) andketotifen-hydrofunarate are also of an interest.

[0295] Sub-dosed antagonists under the points (1) (2) (3) and (4) can becombined. In this case, the dose has to be reduced to 5-50% of the dose,quoted under the points (1)(2)(3) and (4). The strongly sub-dosedantagonist under the points (1)(2)(3) and (4) can be combined withconventional Ca-antagonists and/or with sub-dosed agonists of Gp- andGi-coupled receptors.

(1) Malignancies

[0296] (1) The restoration of the immunocompetence following thesublethal irradiation or high-dose chemotherapy can be accelerated-according to the invention- if cytokines such as M-CSF or GM-CSF arecombined with (a) antagonists of Gs-coupled receptors, (b) agonists ofGi(Gp/Go)-coupled receptors, and/or (c) Ca-antagonists.

[0297] (2) Therapeutic approaches, based on tumor-specific Mabs (andcorresponding immunotoxins), can be improved essentially by combiningthese Mabs (a) with anti-CD3- and/or anti-CD8-Mabs, and (b) with“micro-immunosurgery”, tumor patients are (a) first treated by thecombination of Mab-saving cyclophosphamide plus anti-CD3- oranti-CD8-Mab, then (b) injected by alloreactive donor effector cells(PBM/PBL or T cells) which can be primed against the recipient and (c)treated again by cyclophosphamide plus anti-CD3- or anti-CDB-Mabs inorder to eliminate the alloreactive donor cells, and (d) finallyreinjected with the autologous PBM or T cells, collected before thestart of therapy, to restore the recipient's immunocompetence. In asimplified version, the step (c) can be omitted.

[0298] (4) The reinduction of tumor-specific Ts cells can be prevented(a) by Mabs, directed against immature T cells (e.g. anti-T6-, antiT9-,anti-T10-Mabs), (b) by antagonists of Gs-coupled receptors, and/or (c)by agonists of Gi-coupled receptors.

[0299] (5) To prevent an early RES-elimination of allogeneic effectorcells during the “microimmunosurgery”, a simultaneous injection ofallogeneic erythrocytes or inactivated autologous erythrocytes isrecommended (temporary “RES-blockade”)

[0300] (6) The combination of (a) insuline or antidiabetes with (b)glucose or di- and tricarbonic acids can amplify the above describeddeblocking process of hyperactivated macrophages and T cells. Instead ofdi- and tricarbonic acids, their alkali-salts are recommended, whichlead to the rise of intracellular pHi:

[0301] (7) The combination of (a) amiloride plus plasma-acidifyingagents (e.g. NH₄C1, Iysine.HC1, methionine HC1) with (b) TNF (orTNF-inducers such as LPS) or interferon, augments their ROI-mediatedtarget cell damaging by inhibiting the synthesis of ROI-cleaving enzymes(e.g. SOD).

[0302] (8) Since hyper- or persistently activated effector cells(macrophages, T cells etc.) represent a common element (a) inneiplastic, (b) in autoimmune diseases (c) in atherosclerosis, (d) inbacterial and (retro)viral infections, including HIV, and (e) invaccines, the use of antagonists of Gs-coupled receptors and of agonistsof Gi(Gp)-coupled receptors, as well as ofCa-antagonists/Ca-overload-blockers is recommended in all thesesituations.

[0303] (9) The efficiency of “microimmunosurgery” can be furtherincreased by the in vivo or in vitro sensibilization of donor effectorcells against the recipient (patient). Blastogenic pretransformationconfers the donor effector cells a higher efficacy and a time advantageof 24 days which allows these donor cells to inactivate the patient'spathological subclone(s) before the recipient can organize theanti-donor defense. In this way, the pretreatment of the recipient(patient) by cyclophosphamide plus Mab can be reduced to a minimum.

[0304] (10) Agents, increasing the intracellular cGMP, such as (a)nitrocompounds (e.g. glycerolnitrates, isosorbit-mono- and dinitrates),vasodilators (e.g. minoxidil, (di)hydralazine, Na-nitroprusside) and (c)molsidomine can be used alone or in combination with BRMs, lymphokines,growth factors, methylxanthines (e.g. theophylline), Ca-antagonists orCa-overload-blockers, agonists of Gi(Gp)-coupled receptors, antagonistsof Gs-coupled receptors and/or pHi-increasing or redoxpotential-correcting substances.

[0305] (11) Since vasodilators (e.g. nitrocompounds) and molsidomineinhibit the IP3-mediated Ca²⁺-mobilization, they can be used, alone orcombined with Ca-antagonists, for a rapid deblockade of thosehyperactivated effector cells which show a strongly increased Ca²⁺-leveldue to an excess of signals, as observed e.g. in chronic inflammationand autoimmune disorders. Favorable is the combination withpHi-increasing and/or redox potential correcting agents.

[0306] (12) The combination of molsidomine plus nicergoline (with orwithout cinnarizine) is of a special interest.

[0307] (13) Infection and inflammations can be treated by combiningNSAID with sub-dosed (a) antagonists of Gs-coupled receptors (b)agonists of Gi(Gp)-coupled receptors and/or (c) sub-dosed Ca-antagonists(optimally: Ca- and Na-channels-inhibiting Ca-overload-blockers, such ascinnarizine).

[0308] (14) With the same classes of substances, the in vitro generationof LAK- and TL-cells could be improved essentially.

[0309] (15) The suppressor-monocytes seem to play a crucial negativerole in the process of immunosuppression. Therefore, they have to bedepleted in vivo by anti-Macrophage-Mabs (or immunotoxins), along withthe neutralization of their secretory products (monokins) by Mabs,directed against TNFalpha and IL-1. In addition, themacrophage-stimulating gamma-IFN must be neutralized by the specificMab.

[0310] The effector cells, active in the “microimmunosurgery” process,seem to inactivate both the hyperactivated T cells and thepathologically activated macrophages.

[0311] (16) The cGMP-synthesizing guanylate cyclase (G.C.) can bestimulated, according to the invention, (a) by nitro compounds (“organicnitrates”), such as glycerol-, erythrite- and isosorbite-nitrate, (b) byfurther vasodilators, such as minoxidil, (di)hydralazine,Na-nitroprusside, (c) by compounds such as arginine,N-methyl-D-aspartate(NMDA), L-glutamate, S-acetylthiocholin-iodide,paraquate, D- and L-ornithine, further extreme low concentrations of NOand cb. The most act via stimulation of NO-synthetase (NOS). Themonokines such as TNFalpha and L-1 and lymphokines, such as IFN-gamma(a) switch the cell function from the cell proliferation to the celldifferentiation, and (b) induce the secretion of numerous cytokines viathe H₂O₂-intermediate. Interestingly, the H₂O₂ per se is able to induce,at very low concentrations, the TNF-production. The NO-synthesis goesparallel with the ROI (H₂O₂) -formation and is dependent in its laststep (oxydation of the hydroxylamine-intermediate) on the cooperationwith the compound I (catalase: H₂O₂-complex). The NO is oxydized to N0 ₂and N0 ₃.

[0312] The cGMP-synthesis and mediately the secretion of cytokines canbe achieved also by extremely low concentrations of ROI (e.g. H₂O₂ orMg-peroxide or (NH₄)₂S₂O₄).

[0313] On the other hand, by the inhibition of NO-synthetase or byROI-scavengers and reducing agents (e.g. N-acetyl-cysteine, glutathione,cysteine, ascorbate, penicillamine etc.), the (hyper)production ofcytokines can be inhibited.

[0314] (17) The efficacy of the treatment can be increased by theintratumoral/intralesional application (a) of NOS-activators (b) ofagonists of Gi(Gp)-coupled receptors and/or (c) of antagonists ofGs-coupled receptors.

[0315] (18) Since the cGMP-increasing agents (e.g. nitrocompounds,molsidomine) decrease the Ca_(i)-concentration, the combination of thesecompounds with Ca_(i)- and pHi-increasing agents is recommended. By afine mutual balancing of these substance classes, first the energy ofimmunocompetent cells, caused by hyperactivity, can be broken bynitrocompounds and subsequently the biosynthesis of cytokines and growthfactors can be induced by a parallel Ca_(i)- and pHi-elevation.

[0316] (19) On the other hand, the combination (a) of Cai-reducingstimulators of NOS (b) with agonists of Gs-coupled receptors or withantagonists of Gi(Gp)-coupled receptors and/or (c) with Ca-antagonistsresults in the inactivation of pathologically hyperactivated subclones.This effect can be strengthened by the TNF (or TNF-inducers, like LPS),IFN-gamma and IL-I.

[0317] Of a special interest is also the combination ofCa-overload-blockers, e.g. piperazines (cinnarizine, lidoflazine,flunarizine) with Ca-antagonists (controlled Ca_(i)- and cGMP-rise)

[0318] (20) The use (a) of agonists of Gi(Gp)-coupled receptors, (b) ofantagonists of Gs-coupled receptors,, and/or (c) of NOS-stimulatingcompounds helps to save cytokines and growth, factors. By a fine mutualbalancing, a parallel Ca_(i)-, pHi- and cGMP-rise and herewith a maximalcytokine production can be achieved.

[0319] (21) The pretreatment (“conditioning”) of recipient, i.e. tumorpatient with the cyclophospharnide plus anti-CD3- or anti-CD8-Mab aspart of the “microimmunosurgery” can be replaced by the treatment withthe cyclophosphamide plus anti-T6 (anti-T9; anti-T10; anti-TdT)Mab.

[0320] The combination of cyclophosphamide with the mixture of Mabs,directed against the mature cells (anti-CD3 Mabs) and T cell precursors(e.g. anti-T6-Mabs) is also recommended.

[0321] (22) A combination (a) of antagonists of Gi(Gp)-coupledreceptors, (b) of Ca²+-influx inhibiting Ca-antagonists, (c) ofCa²⁺-mobilization-inhibiting nitrocompounds, (d) of agonists ofGs-coupled receptors (B-sympathomimetics) and/or (e) of alkalizingagents could be lifesaving even during a fatal immune over-reaction(septic or anaphylactic shock). The K-preparations, O₂-scavengers andtolbutarnide or biguanine plus glucose can support this treatment.

[0322] (23) The Ts-cells can be converted to the CTL/Tc-cells (and viceversa). The intracellular ADP/ATP-ratio seems to be critical: a nATP-drop or an ADP-rise seems to signalize the Tc:Ts-interconversion.

[0323] A key role seems to play also the ADP/ATP-dependentcAMP/cGMP-ratio. The AMP, formed by the reaction: 2ADP=ATP+AMP, ispartly dephosphorylated to adenosine which raises, via A2, thepurinergic receptors, the intracellular cAMP-level. The ADP/ATP-decrease(in different stress situations, such as O₂-deficit, physical work,cold, heat) is associated by catecholamine (adrenaline)-rise. Theadrenaline causes also the rise of intracellular cAMP-level. In order toincrease the ADP/ATP-ratio, the following agents are recommended:

[0324] (a) O₂ or O₂-carriers (b) glucose, di- and tricarbonic acids (asalkali-salts), along with antidiabetics (tolbutamide, biguanine etc.),(c) ribose and/or (d) glutamine, glycine and other ATP-precursors. Theadenosine or adrenaline-induced cAMP-rise can be inhibited by thecorresponding receptor blockers.

[0325] (24) The reinduction of tolerance against the tumor cells afterremoval of primary tumor (a) by surgery and/or (b) by chemotherapyand/or (c) by radiotherapy can be inhibited by the antagonists ofGs-coupled receptors and by ligands of Gi(Gp)-coupled receptors. In thecase of B-blockers, the H₂-antihistaminics and A2-purinergic blockers,the ligands are directed from Gs- to Gi(Gp)-coupled receptors.

[0326] (25) The therapy of (a) lymphomas and (b)leukemias of the T-typecan be performed by the “microimmunosurgery” like in solid tumors;again, the sublethal whole body irradiation or high-dose chemotherapy isreplaced by the T cell depletion (by Mab-saving cyclophosphamide plusanti-CD3-Mabs). The transformed T cells are eliminated by allogeneicdonor effector cells. The mature (post-thymus) malignant,cells are MHCII-positive. The immature (pre-thymus) cells are recognized and depletedvia blast-specific structures.

[0327] (26) Autologous BMT in patients with blood cell tumors and thosewith solid tumors (e.g. lung cancer) can be improved by the in vitrotreatment of autologous BM with allogeneic effector cells whose celldeath is preprogrammed. This Ts-depletion on cellular level can becombined with that on humoral level, i.e. with anti-CD8-Mabs and/or withthe depletion of contaminating tumor cells (“purgic”).

[0328] (27) A further elegant technique of deblockade of hyperactivatedeffector cells and of their reactivation in the short opening of thevoltage-operating Ca²⁺-channels (a) by a high frequency (HF)current, or(b) by magnetic induction.

[0329] The HF-currents must be significantly lower than those, used inthe electroporation or electrofusion (Zimmermann). The deblocked orreactivated state can be “frozen” by Ca-antagonists.

[0330] (28) The efficiency of “microimmunosurgery” can be increased bythe combination of Gs- and Gi(Gp)-influencing agents.

[0331] (29) The disappointing efficacy of (a) LAK- and (b)TIL-cells invivo can be explained by the cortisol-depression in plasma. Therefore,(a) the use of adrenostatics (e.g.metopiron), inhibiting the11-B-hydroxylation of glucocorticoids during the LAK- and TIL-infusion,and (b) the admixing of LAK- or TIL-cells, pretransfected in vitro bythe cDNA, encoding either the cortisone-cleaving enzyme20alpha-hydroxysteroid-dehydrogenase (20 alpha SDH) or thecortisone-inactivating enzyme B-glucuronidase, are recommended.

[0332] (30) The efficacy of “microimmunosurgery” can be increased alsoby the pretreatment of the recipient (a) with the ERMs or (b) with thelymphokines (e.g. IFNgamma or IL-4), to render target cells, i.e.pathological subclones more vulnerable.

[0333] (31) The standardized “microimmunosurgery” comprises thefollowing steps: (a) Pooled PBs/PBLs or T cells (from donor A, B, C)whose pathogen-specific subclones can be clonally postexpanded in vitro,e.g. by lectins, are (b) “cell death-preprogrammed” and (c) frozen in amedium (e.g. RPMI1640), containing special cryoprotectants (based on PEGand/or PVP) which warrant the preservation of the preactivated cellstate, in addition to an improved cell viability. (d) Treatment ofpatients, suffering e.g. of cancer or autoimmune disorders is restrictedto the T cell depletion because the “microimmunosurgery” is based on thesimple replacement of the pathologic patient's T set by the healthydonor's T set.

[0334] (32) According to a modified “microimmunosurgery”-procedure, theimmature T- and B-cell precursors, both of the donor (in vitro) and ofthe recipient (patient) are eliminated temporarily by specific Mabs orMabs plus cyclophosphamide; then, the patient's pathological subclonesare eradicated and -as the last step- the depleted precursors arereplaced by autologous or allogeneic (T-depleted) bone marrow. Thespecial advantage is the early exclusion of inducer and transducersuppressor T cells.

[0335] The effector cells can be supported by allogeneic LAK-cells;these can be either premanipulated to express a preprogrammed cell deathor depleted of their T cells (10% of cells). In certain situations, theso pretreated allogeneic LAK-cells can be used alone to eliminate thepathologic subclones.

[0336] (33) Immunocompetent cells, e.g. macrophages express a variety ofadditional, not typically “immunologic” receptors (e.g. for theinsuline, the glucagon, the parathormone etc.) which have been neglectedup to date though they contribute essentially to the activity ofimmunocompetent cells. The therapeutic manipulation of all thesereceptors by agonists and antagonists is subject of this invention.

[0337] (34) Since the early induction of tolerance involves inducer- andtransducer suppressor cells which are both CD4-positive, the use ofanti-VD4-Mabs (or immunotoxins) is recommended for the prevention oftolerance in general. An established tolerance, on the other hand, ismaintained by mature, CD8-positive Ts cells. The same concerns thereinduction of tolerance against the same antigen. In both cases, theuse of anti-CD8-Mabs is recommended. Since immature, CD4-positiveinducer and transducer Ts cells are involved in the reinduction oftolerance, anti-CD4-Mab (alone or combined with anti-CD8-Mab) arerecommended.

[0338] (35) As the IL-2(Tac) receptor (=CD25) is expressed only on(hyper)activated macrophages/monocytes and on the T cells, the paralleldepletion of both pathologically hyperactivated subpopulations can beachieved by anti-CD25-Mabs (or immunotoxins). This procedure can improvethe “microimmunosurgery” (e.g. in the bone marrow and organtransplantation).

[0339] (36) Fibroblasts support the immunosuppressive “tissuerepair”/“wound healing”-function of macrophages and increase e.g. theirPGE2 secretion. Therefore, the neutralization of specific fibroblastgrowth factors, e.g. PDGF, bFGF, FAF, FGTB, PF4 and LTBS is recommended.

[0340] (37) As pathologic processes, such as GvHR, autoreactivity orsarcoidosis, arise from a mutual stimulation of T4 cells andmonocytes/macrophages, the interruption of this activation chain by Mabsagainst IL-I, TNFalpha and/or IFNgamma is recommended.

[0341] (38) Similarly, in BMT the mutual activation cascade, leading toGvH- and HvG-reactivity, has to be interrupted, according to theinvention, by neutralizing the involved cytokine(s) The use of Mabs isfavorable. It is directedagainst the TNFalpha, IL-1, IFNgamma, GM-CSF,M-CSF, IL-6 and IL-4.

[0342] (39) The combating of residual tumor cells and (premicro)metastases, following tumor excision has best chances if the following 2principles are combined: (a) 1^(st) principle: MHC II-expression on thepatient tumor cells, both (al) by the in vitro MHC II-postexpression,induced by IFNgamma, TNFalpha and/or IL-4, and (a2) by the fusion oftumor cells with autologous or allogeneic MHC II-positive cells (b)2^(nd) principle: transfection of patient tumor cells with cytokines,primarily GM-CSF and IL-4. Before their reinfusion, such modified tumorcells can be manipulated to express the preprogrammed cell death. Thelocal cytokine enrichment can be achieved by the addition of allogeneicB cells.

[0343] (40) To increase the humoral response e.g. against HIV, thecombination of antibodies of the IgG- or IgM-isotype with those of theIgE-isotype is recommended. The latter can be constructed by thereplacement of the Fc gamma or Fcu-subunit by the Fc epsilon-subunit.Alternatively, the IgE-percentage in vivo can be increased by thesimultaneous injection of anti-IFNgamma and IL-4(IL-5).

[0344] (41) A long-term incubation of PBMs with lectins (e.g. 1 weekwith ConA or 3 weeks with PHA) leads to a high enrichment ofCD8-positive Ts cells; therefore, the depletion of so selected Ts cells(a) by anti-CD8-Mabs (plus complement) or (b) by alloreactive effectorcells, with preprogrammed cell death, is recommended. The residualPBMs—released of the depression by the Ts cells—are able topost-generate the Tc/CTLs and Th cells.

[0345] In a next procedure, the patient's PBMs are first incubated invitro with lectins (e.g.PHA) and thereafter treated by increasingconcentrations of anti-CD8-Mab (+complement), until the onset of the Tc-and Th-deblockade. Among the deblocked Tc- and Th-cells, tumor-specificclones can be expected; after a further clonal expansion, thesetumoricidal subclones can be reinjected into the patient.

[0346] (42) During their maturation process, the erythrocytes lose thenuclea. Therefore, the ennucleated precursors (reticulocytes) are of aspecial interest as carriers of temporarily limited functions (e.g. invivo production of cytokines, Mabs and suppressor factors or localsource of irradiation). In these cells, the cell death need not bepreprogrammed.

[0347] (43) The controlled DNA-damaging described above (“cell deathpreprogramming”) can be carried out by DNA-selective cytotoxic agents,primarily vinca alkaloids, bleomycin, ICRF-159, busulfan, DDP, VP16231(EPE) and EPT, in addition to the above mentioned mitomycinc. Thecontrolled DNA-cross-linking can be achieved by so called “bifunctionalalkylating agents”.

[0348] According to a further aspect of the invention, the use of thecombination of a Ca-antagonist plus an agent, decreasing thecAMP/cGMP-ratio is recommended as the drug for the treatment of cancer,viral and bacterial infections, as well as autoimmune disorders.

[0349] In addition, the use of a combination, consisting of an agent,eliminating hyperactivated effector cells, and of alloreactive cellswith preprogrammed cell death is recommended as the drug for thetreatment of cancer, viral infections and autoimmune diseases.

[0350] According to the invention, an additional approach in thetreatment of cancer, viral infections and autoimmune disorders, based onthe elimination or down-regulation of immunological effector cells,enables the patient's own immune system to restore the pre-diseasestate.

SUMMARY

[0351] A drug, affecting the hyperactivated.immunologic effector cells,comprising (I) a Ca-antagonist and (II) an agent, reducing theintracellular cAMP/cGMP-ratio, is being described. In addition, a drug,affecting the hyperactivated immunologic effector cells, consisting (I)of an agent, eliminating the hyperactivated effector cells and (II) ofalloreactive cells with predetermined cell death, is being described aswell.

1. Drugs, affecting hyperactivated immunologic effector cells,consisting of (I) a Ca-antagonist, and (II) an agent, reducing theintracellular cAMP/cGMP-ratio.
 2. Drugs, according to the claim 1,specified by the fact that Ca-antagonist is a Ca-overload-blocker. 3.Drugs, according to the claim 1 or 2, specified by the fact thatCa-antagonist is-cinnarizine.
 4. Drugs, according to one of thepreceding claims, specified by the fact that component (II) is a drugwhich decreases the cAMP-level in the cell and/or a drug which increasesthe cGMP-level.
 5. Drugs, according to the claim 4, specified by thefact that the cAMP level-decreasing drug is an antagonist of Gs-coupledreceptors and/or an antagonist of Gi/Gp/Go-coupled receptors.
 6. Drugs,according to the claims 4 or 5, specified by the fact that component(II) is an antagonist of B-adrenergic receptors, histamine receptorsand/or A2-purinergic receptors.
 7. Drugs, according to the claim 6,specified by the fact that the antagonist of B-adrenergic receptors ispropranolol.
 8. Drugs, affecting hyperactivated immunologic effectorcells, comprising (I) an agent, eliminating hyperactivated effectorcells, and (II) alloreactive cells with a preprogrammed cell death. 9.Drugs, according to the claim 8, specified by the fact that component(I) is a T cell-depleting agent.
 10. Drugs, according to the claim 9,specified by the fact that complement (I) comprises antibodies, directedagainst CD8 and/or CD3.
 11. Drugs, according to one of the claims 8 to10, specified by the fact that component(I) contains in addition acytotoxic agent, especially cyclophosphamide.
 12. Drugs, according tothe claims 8 to 11, specified by the fact that component (II) comprisesallogeneic cells whose DNA was manipulated in a controlled way and whichare able to inactivate the pathologic Ts cells and/or hyperactivatedmacrophages.
 13. The use of a combination of a Ca-antagonist and a drug,able to reduce the intracellular cAMP/cGMP-ratio, to produce a drug forthe treatment of cancer, viral and bacterial infections and autoimmunedisorders.
 14. The use of a combination, consisting of an agent,eliminating the hyperactivated effector cells, and of alloreactive cellswith the preprogrammed cell death to produce a drug for the treatment ofcancer, viral infections and autoimmune diseases.
 15. Procedure for thetreatment of cancer, viral infections and autoimmune disorders, based onthe elimination of hyperactivated immunological effector cells or theirdown-regulation to the normal state which enables the patient's immuneresponse to restore the pre-disease state.